Methods and compositions of localizing nucleic acids to arrays

ABSTRACT

Methods and compositions are disclosed relating to the localization of nucleic acids to arrays such as silane-free arrays, and of sequencing the nucleic acids localized thereby.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/707,527 filed on Dec. 9, 2019 which is a continuation of U.S.application Ser. No. 15/864,384 filed on Jan. 8, 2018 which issued asU.S. Pat. No. 10,525,437 on Jan. 7, 2020 which is a continuation of U.S.application Ser. No. 15/162,304 filed on May 23, 2016 which issued asU.S. Pat. No. 9,889,422 on Feb. 13, 2018 which is a continuation of U.S.application Ser. No. 14/592,766 filed on Jan. 8, 2015 which issued asU.S. Pat. No. 9,376,710 on Jun. 28, 2016 which is a continuation of U.S.application Ser. No. 14/053,333 filed on Oct. 14, 2013 which issued asU.S. Pat. No. 8,969,258 on Mar. 3, 2015, which is a divisional of U.S.application Ser. No. 13/548,558 filed on Jul. 13, 2012 which issued asU.S. Pat. No. 8,563,477 on Oct. 22, 2013, which is a continuation ofU.S. application Ser. No. 10/585,373 filed on Oct. 20, 2008 which is theU.S. National Stage Application of PCT Application No. PCT/GB2005/000033filed on Jan. 7, 2005, which claims priority from Great BritainApplication Serial No. 0400253.1 filed on Jan. 7, 2004 and EuropeanApplication Serial No. 04254726.5 filed on Aug. 5, 2004, the entiredisclosures of which are incorporated herein by reference.

SEQUENCE LISTING

The present application includes a sequence listing in Electronicformat. The Sequence Listing is provided as a file entitledILLINC214C6SEQLIST, created Mar. 17, 2021, which is approximately 4 kbin size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to the construction of arrays of molecules. Inparticular, the invention relates to the preparation of a hydrogelsurface useful in the formation and manipulation of arrays of molecules,particularly polynucleotides and to the chemical modification of theseand other arrays.

Description of the Related Art

Advances in the study of molecules have been led, in part, byimprovement in technologies used to characterize the molecules or theirbiological reactions. In particular, the study of nucleic acids, such asDNA and RNA, and other large biological molecules, such as proteins, hasbenefited from developing technologies used for sequence analysis andthe study of hybridisation events.

An example of the technologies that have improved the study of nucleicacids is the development of fabricated arrays of immobilised nucleicacids. These arrays typically consist of a high-density matrix ofpolynucleotides immobilised onto a solid support material. Fodor et al.,Trends in Biotechnology (1994) 12:19-26, describe ways of assembling thenucleic acid arrays using a chemically sensitised glass surfaceprotected by a mask, but exposed at defined areas to allow attachment ofsuitably modified nucleotides. Typically, these arrays may be describedas “many molecule” arrays, as distinct regions are formed on the solidsupport comprising a high density of one specific type ofpolynucleotide.

An alternative approach is described by Schena et al., Science (1995)270:467-470, where samples of DNA are positioned at predetermined siteson a glass, microscope slide by robotic micropipetting techniques.

A further development in array technology is the attachment of thepolynucleotides to a solid support material to form single moleculearrays (SMAs). Arrays of this type are disclosed in WO00/06770. Theadvantage of these arrays is that reactions can be monitored at thesingle molecule level and information on large numbers of singlemolecules can be collated from a single reaction.

Although these arrays offer particular advantages in sequencingexperiments, the preparation of arrays at the single molecule level ismore difficult than at the multi-molecule level, where losses of targetpolynucleotide can be tolerated due to the multiplicity of the array.Moreover, where the sequence of a polynucleotide is determined bysequential incorporations of labelled nucleotides, a further problemwhich arises is the occurrence of non-specific binding of nucleotides tothe array, for example to the surface of the array. There is, therefore,a constant need for improvements in the preparation of arrays ofmolecules, particularly polynucleotides, for example single moleculearrays of polynucleotides, for sequencing procedures.

Solid-supported molecular arrays have been generated previously in avariety of ways. Indeed, the attachment of biomolecules (such asproteins and nucleic acids, e.g. DNA) to a variety ofsupports/substrates (e.g. silica-based substrates such as glass orplastics or metals) underpins modern microarray and biosensortechnologies employed for genotyping, gene expression analysis andbiological detection.

In nearly all examples where biomolecules have been immobilised on solidsupports, the attachment chemistry is designed around the support. Forexample, silanes (e.g. functionalised silanes such as chloro- oralkoxy-silanes) are commonly used to modify glass; thiols are often usedto modify the surface of gold. A potential problem here is that theagents used to modify one surface are often unsuitable for modifying thesurface of another support. For example, thiols cannot be used to modifyglass, nor can silanes be used to modify gold.

Silica-based substrates such as silica or glass are often employed assupports on which molecular arrays are constructed. It would bedesirable to be able to use chemistry useful in modifying such supportswith other supports.

Prior to the construction of any silica-based solid-supported arrays,the support surface is generally thoroughly cleaned. With silica-basedsubstrates, the resultant cleaned surface possesses hydroxyl groupswhich are either neutral and/or deprotonated and thus negativelycharged. As a result there is a degree of resistance to non-specificbinding of nucleotides used in sequencing experiments. Either theneutral hydroxyl groups do not attract the negatively chargednucleotides, or the deprotonated groups' negative charge serves to repelthe nucleotides. Regardless, the effect of the surface towards thenon-specific, and undesired, binding of nucleotides is not high and itis desirable to lessen the extent of non-specific binding in sequencingexperiments. This serves to reduce background “noise” during thedetection of each individual nucleotide in each step in sequencingexperiments.

Another way in which polynucleotides (and other molecules) have beendisplayed previously on the surface of solid support is through the useof hydrogels. Molecular arrays, e.g. microarrays, of molecules,particularly polynucleotides, are of use in techniques including nucleicacid amplification and sequencing methods. In preparing hydrogel-basedsolid-supported molecular arrays, a hydrogel is formed and moleculesdisplayed from it. These two features—formation of the hydrogel andconstruction of the array—may be effected sequentially orsimultaneously.

Where the hydrogel is formed prior to formation of the array, it istypically produced by allowing a mixture of comonomers to polymerise.Generally, the mixture of comonomers contain acrylamide and one or morecomonomers, the latter of which permit, in part, subsequentimmobilisation of molecules of interest so as to form the moleculararray.

The comonomers used to create the hydrogel typically contain afunctionality that serves to participate in crosslinking of the hydrogeland/or immobilise the hydrogel to the solid support and facilitateassociation with the target molecules of interest.

Generally, as is known in the art, polyacrylamide hydrogels are producedas thin sheets upon polymerisation of aqueous solutions of acrylamidesolution. A multiply unsaturated (polyunsaturated) crosslinking agent(such as bisacrylamide) is generally present; the ratio of acrylamide tobisacrylamide is generally about 19:1. Such casting methods are wellknown in the art (see for example Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, 3^(rd) Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor Laboratory Press, NY) and need not bediscussed in detail here.

As an alternative to the use of hydrogel-supported molecular arrays, theuse of polyelectrolyte multilayers (PEMs) has been reported (E. P.Kartov et al., Biotechniques (March 2003), 34:505-510; and I. Braslayskyet al., Proct Nat. Acad. Sci (1 Apr. 2003), 100 (7), 3960-3964) to allowsequencing experiments to be conducted in which fluorescently labelledmolecules are incorporated into DNA strands and then identified byfluorescence microscopy. The authors report that, by using PEMs, thecharge density on the surface may be tuned so as to repel labellednucleotides selectively by constructing the PEMs such that the finallayer bears a negative charge.

Accordingly, the authors describe such a PEM which, after itsconstruction, was used in the formation of a molecular array. The latterwas formed initially by biotinylating the surface using a commerciallyavailable kit (EZ-Link™ kit from Pierce Chemical (Rockford, Ill., USA)).The biotinylated PEM was then coated with Streptavidin-Plus™ (Prozyme,San Leandro, Calif., USA) to which biotinylated DNA was attached. Inthis way the biotinylated DNA is attached to covalently bound biotinthrough specific noncovalent interactions to “sandwiched” streptavidinmolecules.

The authors of B. P. Kartov et al. (infra) and I. Braslaysky et al. (twoauthors are common to both publications) report that the final,negatively charged, polyacrylic acid layer is intended to preventnegatively charged labelled nucleotides binding to the surface. It isclear, however, that this was not successful in every instance since itis reported in I. Braslaysky et al. (infra) that the identity of thethird or fourth incorporated nucleotide could not be determined (was“ambiguous”). According to the authors, this was caused by “increasingnon-specific binding of unincorporated nucleotides”.

Accordingly, there exists a need for a method of providing arrays ofmolecules, particularly polynucleotides, which arrays have a lessertendency to interact nonspecifically with other molecules (and inparticular (optionally fluorescently labelled) nucleotides used insequencing experiments) than those available in the prior art. There isalso a need for a general method for modifying a solid support to allowthe preparation of supports useful in the preparation of arrays.

SUMMARY OF THE INVENTION

Heretofore, some form of covalent surface modification of the solidsupport has been practised in order to achieve satisfactoryimmobilisation of either hydrogel-based molecular arrays or hydrogels towhich it is desired to array molecules. Surprisingly, we have found thatsuch functional modification of the support is not necessary in order toachieve satisfactory immobilisation of arrays of molecules of interest,in particular polynucleotides. In order to make useful supported arrayscapable of binding molecules of interest, we have found that a mixtureof comonomers comprising at least one hydrophilic monomer and afunctionalised comonomer (functionalised to the extent that the monomeronce incorporated into the polymer is capable of binding the molecule ofinterest to the surface of the hydrogel) may be polymerised so as toform a hydrogel capable of being immobilised on a solid supported,preferably a silica-based, substrate.

Viewed from one aspect, therefore, the invention provides a method ofpreparing a hydrogel immobilised to a solid support comprisingpolymerising on said support a mixture of:

(i) a first comonomer which is acrylamide, methacrylamide, hydroxyethylmethacrylate or N-vinyl pyrrolidinone; and

(ii) a second comonomer which is a functionalized acrylamide or acrylateof formula (I):

H₂C═C(H)—C(═O)-A-B—C  (I);

or a methacrylate or methacrylamide of formula (II):

or H₂C═C(CH₃)—C(═O)-A-B—C—  (II)

(wherein:

A is NR or O, wherein R is hydrogen or an optionally substitutedsaturated hydrocarbyl group comprising 1 to 5 carbon atoms;

—B— is an optionally substituted alkylene biradical of formula—(CH₂)_(n)— wherein n is an integer from 1 to 50; and wherein n=2 ormore, one or more optionally substituted ethylene biradicals —CH₂CH₂— ofsaid alkylene biradical may be independently replaced by ethenylene andethynylene moieties; and wherein n=1 or more, one or more methylenebiradicals —CH₂— may be replaced independently with an optionallysubstituted mono- or polycyclic hydrocarbon biradical comprising from 4to 50 carbon atoms, or a corresponding heteromonocyclic orheteropolycyclic biradical wherein at least 1 CH₂ or CH₂ is substitutedby an oxygen sulfur or nitrogen atom or an NH group; and

C is a group for reaction with a compound to bind said compoundcovalently to said hydrogel) to form a polymerized product,

characterised in that said method is conducted on, and immobilises thepolymerised product to, said support which is not covalentlysurface-modified.

Viewed from a second aspect, the invention provides a solid-supportedhydrogel obtainable according to the method of the first invention.

Viewed from a third aspect, the invention provides a method of preparinga solid-supported hydrogel-based molecular array by attaching one ormore molecules of interest to reactive sites present in thesolid-supported hydrogel according to the invention.

In a particular embodiment the invention provides a method of preparinga solid-supported hydrogel-based molecular array which is a clusteredarray by attaching oligonucleotide primers to reactive sites present inthe solid-supported hydrogel and performing nucleic acid amplificationof a template using the bound primers.

Viewed from a fourth aspect, the invention provides a solid-supportedhydrogel-based molecular array obtainable according to the third aspectof the invention.

Viewed from a fifth aspect, the invention provides the use of amolecular array according to the fourth aspect of the invention in anymethod of analysis which requires interrogation of the molecules ofinterest or molecules bound thereto.

The use of solid-supported hydrogel arrays in single molecule arrayapplications has not been conducted previously. Thus viewed from a sixthaspect, the invention provides the use of solid-supported hydrogelarrays in single molecule array applications, preferably wherein saidarrays are obtainable, and generally obtained, by a method comprising:

(1) preparing a hydrogel immobilised to a solid support comprisingpolymerising on said support a mixture of:

(i) a first comonomer which is acrylamide, methacrylamide, hydroxyethylmethacrylate or N-vinyl pyrrolidinone; and

(ii) a second comonomer which is a functionalized acrylamide or acrylateof formula (I):

H₂C═C(H)—C(═O)-A-B—C  (I);

or a methacrylate or methacrylamide of formula (II):

or H₂C═C(CH₃)—C(═O)-A-B—C—  (II)

(wherein:

A is NR or O, wherein R is hydrogen or an optionally substitutedsaturated hydrocarbyl group comprising 1 to 5 carbon atoms;

—B— is an optionally substituted alkylene biradical of formula—(CH₂)_(n)— wherein n is an integer from 1 to 50; and

wherein n=2 or more, one or more optionally substituted ethylenebiradicals —CH₂CH₂— of said alkylene biradical may be independentlyreplaced by ethenylene and ethynylene moieties; and wherein n=1 or more,one or more methylene biradicals —CH₂— may be replaced independentlywith an optionally substituted mono- or polycyclic hydrocarbon biradicalcomprising from 4 to 50 carbon atoms, or a correspondingheteromonocyclic or heteropolycyclic biradical wherein at least 1 CH₂ orCH₂ is substituted by an oxygen sulfur or nitrogen atom or an NH group;and

C is a group for reaction with a compound to bind said compoundcovalently to said hydrogel) to form a polymerized product, and

(2) attaching one or more molecules of interest to reactive sitespresent in the hydrogel produced in step (1).

The invention also provides the use of, and methods of using, arrays,preferably single molecule arrays according to invention, in theinterrogation of the molecules in said array.

One of the advantages of the hydrogel-based molecular arrays andhydrogels of the invention, it has been found, is that omission of acovalent surface-modification step (particularly of the solid support)affords a surface having greater passivity than in the prior art,particularly when compared to those instances where the use of thesilane-modifying agents described above with silica-based substrates areemployed.

The provision of a surface which leads to as little as possibleaspecific surface contamination is clearly advantageous where thehydrogels are used to construct arrays, such as microarrays, andpreferably clustered arrays or SMAs, to be used in sequencing reactions.

Notwithstanding this, however, the hydrogels of this invention havefunctionality used in forming, or reacting with, the molecules which arearrayed. Consequentially, these hydrogels suffer too, albeit to a morelimited extent than prior art hydrogels supported upon a functionallymodified support, from a degree of aspecific nucleotide binding duringsequencing.

Surprisingly, we have found that the solid-supported hydrogel-basedmolecular arrays of the invention may be still further improved byeffecting certain modifications to these arrays after their formationbut before initiation of any manipulation of, e.g. interrogation of, themolecules in the array. These arrays are of even greateradvantageousness in, for example, polynucleotide sequencing reactionsbecause the surfaces of the arrays may be rendered more passive, andthus less reactive, towards molecules such as optionally labellednucleotides.

Accordingly the method according to the third aspect of the inventionpreferably contains the additional step of applying to the array soproduced polyelectrolyte or neutral polymers. This improvement is ofcorresponding benefit to the other aspects of the invention directed tothe arrays themselves, and the uses, and methods of using, such arrays.

The improvement to the solid-supported hydrogel-based molecular arrays,and uses of the arrays, of the invention is of general utility in thepreparation and use of molecular arrays. It will be appreciated from theforegoing discussion that, in the preparation of arrays of molecules todate, particularly in the preparation of arrays of polynucleotides,these have invariably been assembled by initial preparation of thesupport, whether this be achieved by modification of a silica-basedsubstrate, or formation of a PEM on a glass substrate, or formation of ahydrogel on glass or other solid supports. Only once the constitution ofthe solid support has been finalised is the array formed by reactionbetween the support and the molecules of interest. The array is thenused, without further modification, in methods of analysis such assequencing experiments whereby the molecules, typically polynucleotides,are interrogated.

Viewed from a still further aspect, therefore, the invention provides amethod of modifying a molecular array, which molecular array comprises aplurality of molecules of interest, preferably biomolecules, immobilisedto a surface of a support, said method comprising the step of applyingto the array polyelectrolyte or neutral polymers.

Viewed from a still further aspect, the invention provides a moleculararray obtainable according to the immediately preceding aspect of theinvention.

Viewed from a still further aspect, the invention provides the use of amolecular array according to the immediately preceding aspect of theinvention in any method of analysis which requires interrogation of theimmobilized biomolecules, or of molecules bound thereto.

The present invention will now be further described. In the followingpassages different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images which show the detection of fluorescence fromoligonucleotides immobilised on substrates both according to and notaccording to the invention, in accordance with Example 11. The improvedbinding of phosphorothioate-terminated DNA (PS-DNA) overhydroxyl-terminated DNA may be seen;

FIG. 2 shows the relative levels of positive signal due to PS-DNAbinding versus negative noise from OH-DNA binding resultant fromcoupling of 1 μM PS-DNA and 1 μM OH-DNA to plastic and fused silica(SPECTRASIL® glass) detected on the surfaces whose preparation isdescribed in Example 10;

FIG. 3 shows the apparent stability of the specifically adsorbed PS-DNA(in 50 mM phosphate buffer (pH7, 65° C.)) for various plastics materialsis approximately the same as that for SPECTRASIL™ glass;

FIG. 4 shows images of the detection of fluorescence fromoligonucleotides, immobilised on substrates both according to and notaccording to the invention, in accordance with Example 13. The improvedbinding of phosphorothioate-terminated DNA (PS-DNA) overhydroxyl-terminated DNA may be seen.

FIG. 5 is a schematic illustration of a prior art method of solid-phaseamplification in which a mixture of oligonucleotide primers and templatestrands are simultaneously grafted onto a solid support.

FIG. 6 is a schematic illustration of a method of solid-phaseamplification according to the invention in which oligonucleotideprimers are first grafted onto a solid support and then hybridised totemplate strands.

FIG. 7 illustrates the use of spacer nucleotides to improve efficiencyof hybridisation between an immobilised polynucleotide primer and alabelled target, according to Example 14.

FIGS. 8(a) and 8(b) show the results of hybridisation experimentscarried out in microtiter plates between various concentrations ofimmobilised primers—with and without spacer nucleotides—and targetoligonucleotides labelled with Texas red (TXR). In FIG. 8(a) theidentity of the immobilised primer is indicated along the top of theplate. Templates were added in groups of four wells, as shown in thekey. Hybridisation was carried out in two different buffers—a PCR buffer(PCR) and 5×SSC (SSC). Each immobilised primer was tested forhybridisation with a complementary primer (i.e. P5′ is complementary toP5) and with a control non-complementary primer. FIG. 8(b) illustratestypical results in graphical form.

FIG. 9 illustrates experimental set-up for hybridisation experimentsdescribed in Example 14.

FIG. 10 illustrates experimental set-up and results of hybridisationexperiments using various immobilised oligonucleotide primers, with andwithout spacer nucleotides, and complementary labelled targetoligonucleotides.

FIG. 11 graphically represents the results of typical hybridisationexperiments according to Example 14.

FIGS. 12(a) and 12(b) illustrate the results of hybridisationexperiments using immobilised oligonucleotide primers containing varyingnumbers of spacer nucleotides and complementary labelled targetoligonucleotides. FIG. 12(a) shows experimental set-up and results. FIG.12(b) provides a graphical representation of the results of typicalhybridisation experiments for varying spacers versus P5′ target. FIG.12(c) provides a similar representation versus P7′ target.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention, as described and claimed herein, provides an improvedmethod for displaying molecules of interest, and particularlybiomolecules (biological molecules) such as polynucleotides and proteins(preferably polynucleotides) displayed on the surface of a solidsupport, preferably a solid-supported hydrogel.

The solid upon which the hydrogel is supported is not limited to aparticular matrix or substrate. Indeed, this is one of the advantages ofthe invention: the same chemistry used to modify silica-based substratescan be applied to other solid supports and allows the solid support tobe adapted to suit any particular application to which it is desired tobe put rather than being constrained by the surface chemistry it ispossible to perform on any given support. Solids which may be of use inthe practise of this invention thus include silica-based substrates,such as glass, fused silica and other silica-containing materials; theymay also be silicone hydrides or plastic materials such as polyethylene,polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters,polycarbonates and poly (methyl methacrylate). Preferred plasticsmaterial are poly (methyl methacrylate), polystyrene and cyclic olefinpolymer substrates. Alternatively, other solid supports may be used suchas gold, titanium dioxide, or silicon supports. The foregoing lists areintended to be illustrative of, but not limited to, the invention.Preferably, the support is a silica-based material or plastics materialsuch as discussed herein.

Advantages in using plastics-based substrates in the preparation and useof molecular arrays include cost: the preparation of appropriateplastics-based substrates by, for example injection-moulding, isgenerally cheaper than the preparation, e.g. by etching and bonding, ofsilica-based substrates. Another advantage is the nearly limitlessvariety of plastics allowing fine-tuning of the optical properties ofthe support to suit the application for which it is intended or to whichit may be put.

Where metals are used as substrates, this may be because of the desiredapplication: the conductivity of metals can allow modulation of theelectric field in DNA-based sensors. In this way, DNA mismatchdiscrimination may be enhanced, the orientation of immobilisedoligonucleotide molecules can be affected, or DNA kinetics can beaccelerated.

Preferably the support is silica-based but the shape of the supportemployed may be varied in accordance with the application for which theinvention is practiced. Generally, however, slides of support material,such as silica, e.g. fused silica, are of particular utility in thepreparation and subsequent interrogation of molecules. Of particular usein the practice of the invention are fused silica slides sold under thetrade name SPECTRASIL™. This notwithstanding, it will be evident to theskilled person that the invention is equally applicable to otherpresentations of solid support (including silica-based supports), suchas beads, rods and the like.

The genesis of the invention is the recognition by the inventors thatthe surface of the support need not be covalently modified in order fora hydrogel to be immobilised thereto. As described herein, the step ofcovalent surface modification may be omitted when the comonomer mixturedescribed and claimed herein is used to produce a hydrogel.

If it is desired to display molecules of interest, e.g. biomolecules,these may be any biological molecule which it is desired to analyse. Ofparticular interest are polypeptides or proteins (including enzymes) andpolynucleotides, with polynucleotides being particularly preferred.

As used herein, the term “polynucleotide” refers to nucleic acids ingeneral, including DNA (e.g. genomic DNA cDNA), RNA (e.g. mRNA),synthetic oligonucleotides and synthetic nucleic acid analogs.Polynucleotides may include natural or non-natural bases, orcombinations thereof and natural or non-natural backbone linkages, e.g.phosphorothioates, PNA or 2′-O-methyl-RNA, or combinations thereof.

Whilst it will be appreciated that the solid-supported hydrogels of theinvention are useful for the presentation of many different types ofmolecules, the hydrogels are of particular use in the formation ofarrays of polynucleotides and their subsequent analysis. For thisreason, the majority of the subsequent discussion will focus upon theutility of the supported hydrogels of the invention in the preparationof polynucleotide arrays (both single molecule arrays and microarrays,such as clustered arrays formed by nucleic acid amplification) althoughit is to be understood that such applications in no way limit theinvention. Moreover, since silica-based supports are typically used tosupport hydrogels and hydrogel arrays, the subsequent discussion willfocus on the use of silica-based supports. Again, this is not to beconsidered as a limitation of the invention; rather this isdemonstrative of a particular advantage of the invention for improvingprocedures directed to constructing arrays of molecules onsilica-supported hydrogels. The improvement offered will be evident froma review of the prior art.

WO00/31148 discloses polyacrylamide hydrogels and polyacrylamidehydrogel-based arrays in which a so-called polyacrylamide prepolymer isformed, preferably from acrylamide and an acrylic acid or an acrylicacid derivative containing a vinyl group. Crosslinking of the prepolymermay then be effected. The hydrogels so produced are solid-supported,preferably on glass. Functionalisation of the solid-supported hydrogelmay also be effected.

WO01/01143 describes technology similar to WO00/31148 but differing inthat the hydrogel bears functionality capable of participating in a[2+2] photocycloaddition reaction with a biomolecule so as to formimmobilised arrays of such biomolecules. Dimethylmaleimide (DMI) is aparticularly preferred functionality. The use of [2+2]photocycloaddition reactions, in the context of polyacrylamide-basedmicroarray technology is also described in WO02/12566 and WO03/014392.

U.S. Pat. No. 6,465,178 discloses the use of reagent compositions inproviding activated slides for use in preparing microarrays of nucleicacids; the reagent compositions include acrylamide copolymers. Theactivated slides are stated to be particularly well suited to replaceconventional (e.g. silylated) glass slides in the preparation ofmicroarrays.

WO00/53812 discloses the preparation of polyacrylamide-based hydrogelarrays of DNA and the use of these arrays in replica amplification.

None of the prior art described herein discloses the preparation of asolid-supported hydrogel wherein the solid support is not covalentlymodified.

Once hydrogels have been formed, molecules may then be attached to themso as to produce molecular-arrays, if desired. Attachment has beeneffected in different ways in the prior art. For example, U.S. Pat. No.6,372,813 teaches immobilisation of polynucleotides bearingdimethylmaleimide groups to the hydrogels produced which beardimethylmaleimide groups by conducting a [2+2] photocycloaddition stepbetween two dimethylmaleimide groups—one attached to the polynucleotideto be immobilised and one pendant from the hydrogel.

Where the molecular array is formed after generation of the hydrogel,two strategies have been employed to achieve this end. Firstly, thehydrogel may be modified chemically after it is produced. Problems withthis approach include an overall low efficiency in the preparation ofthe array and the low stability relating to the attachment chemistry,particularly upon exposure to high temperatures, ionic solutions andmultiple wash steps.

A more common alternative is to effect polymerisation with a comonomerhaving a functionality primed or pre-activated to react with themolecules to be arrayed.

Alternatives to initial formation of hydrogels followed by subsequentarraying of molecules thereto have been described in the prior art wherethe array is formed at the same time as the hydrogel is produced. Thismay be effected by, for example, direct copolymerisation ofacrylamide-derivatized polynucleotides. An example of this approach isdescribed in WO01/62982 in which acrylamide-derivatized polynucleotidesare mixed with solutions of acrylamide and polymerisation is effecteddirectly.

Mosaic Technologies (Boston, Mass., USA) produce ACRYDITE™ (anacrylamide phosphoramidite) which can. be reacted with polynucleotidesprior to copolymerisation of the resultant monomer with acrylamide.

Efimov et al. (Nucleic Acids Research, 1999, 27 (22), 4416-4426)disclose a further example of a simultaneous formation of hydrogel/arrayin which copolymerisation of acrylamide, reactive acrylic acidderivatives and the modified polynucleotides having 5′- or 3′-terminalacrylamide groups is effected.

The above-described techniques, however, in which the hydrogel isgenerated simultaneously with the array by introduction of appropriatecomonomers bearing the molecules of interest, suffer from problemsincluding damage to the molecules of interest during polymerisation.

A variety of solid supports have been used in the prior art to generatehydrogel-based solid-supported molecular arrays. These include thosesupports discussed earlier. Generally the preferred solid supportcomprises a silica-based substrate. Examples of silica-based substratesinclude fused silica and glass.

To the best of our knowledge, in all instances of silica-based supportedhydrogels described in the prior art the silica is chemically modifiedin some way so as to attach covalently a chemically reactive groupcapable of reacting with either the hydrogel or a partially formedhydrogel (e.g. a prepolymer (PRP)). The surface-activating agent istypically an organosilicon (organosilane) compound. Most commonly, it isγ-methacryloxypropyltrimethoxysilane, known as “Bind Silane” or“Crosslink Silane” and commercially available from Pharmacia, althoughother silicon-based surface-activating agents are also known, such asmonoethoxydimethylsilylbutanal, 3-mercaptopropyl-trimethoxysilane and3-aminopropyltrimethoxysilane (all available from Aldrich). In this way,pendant functional groups such as amine groups, sulfhydryl groups,aldehyde groups or polymerisable groups (e.g. olefins) may be attachedto the silica.

It will be clear from the preceding discussion that the invention isparticularly useful when the solid support used is silica-based sincethe silica-based support need not be covalently modified by itspreactivation with a silylating agent as described above in order toimmobilise the hydrogel thereto. Clearly, however, the polymersdescribed herein may still be (in certain aspects of the invention)attached to silica-based supports which have been surface-activated,e.g. with an organosilane molecule as described above; the fulladvantages of the invention will, however, not be obtained in this way.They may also be, of course, attached to the other solid supportsdisclosed herein, in particular plastics such as poly(methylmethacrylate) and polyolefins. Such plastics are readily availablecommercially, e.g. from Arnie, Corning, Zeon Chemical Ltd and others.

The surface-modification of silica-based solid supports by means otherthan covalent attachment of an organosilicon moiety is not excluded fromthe scope of this invention. Preferably, however, no activation of thesilica—by covalent modification of a surface thereof or by any othermeans—is effected prior to effecting polymerisation thereon.

It will be understood that the terms such as “covalentsurface-modification”, “covalent surface-modifying” and “covalentsurface-modified” do not embrace the simple cleaning and/or drying ofsubstrates, particularly silica substrates, prior to their use.Generally such steps will be conducted prior to any polymerisation butdo not constitute a surface-modifying step since no covalentmodification of a surface is essentially effected by such steps.

Where the substrate is silica-based, cleaning will be achieved bycontact with one or more organic solvents such as acetone, isopropanol(IPA), ethanol and the like, or with water or aqueous acidic or alkalinesolutions such as dilute hydrochloric or sulphuric acids or dilutesodium hydroxide. Silica-based supports may be also cleaned by contactwith a detergent solution (e.g. Decon 90). These various cleaning stepsmay be conducted individually or in combination, e.g. sequentially.Drying may be effected, for example, by heating of silica slides attemperatures of from 40° C. to 200° C., preferably 80° C. to 150° C. forbetween 5 minutes to 24 hours, preferably at temperatures of around 120°C., and preferably for 1 to 2 hours.

The cleaning and drying steps described above will be and are understoodby those skilled in the art not to constitute any form of covalentsurface modification (and in particular no form of covalent surfacemodification in which an organosilicon (organosilane) moiety isattached). Such steps serve to effect removal of surface contamination(e.g. dirt or dust) and will generally be conducted prior to use ofsilica-based materials in most scientific applications. Heating of glassto very high temperatures (e.g. 1000° C. or higher or even 300° C. orhigher) or by contact with materials known to dissolve, or etch glass(such as hydrofluoric acid), whilst unlikely to be conducted in thecleaning of silica-based substrates in advance of practice of thisinvention, is not to be considered as constituting a form of surfacemodification.

For substrates that are not silica-based other cleaning techniques maybe appropriate, as will be apparent to those skilled in the art. Forexample, plastic substrates may be cleaned by contact with (e.g.immersion in) any convenient detergent (Decon 90 is an example) followedby thorough rinsing with water, preferably purified water such asMilliQ, prior to drying.

The methods by which the mixture of comonomers are polymerised in theinvention are not characteristic of this invention and will be known tothe skilled person (e.g. by recourse to Sambrook et al. (supra).Generally, however, the polymerisation will be conducted in an aqueousmedium, and polymerisation initiated by any suitable initiator.Potassium or ammonium persulfate as an initiator is typically employed.Tetramethylethylenediamine (TMEDA or TEMED) may be and generally is usedto accelerate the polymerisation.

It is important to note that, in contrast to the teaching of hydrogelpreparation in the prior art concerned with the preparation of moleculararrays, it is not necessary that a polyunsaturated crosslinking agentsuch as bisacrylamide or pentaerythritol tetraacrylate is present in themixture which is polymerised; nor is it necessary to form PRP-typeintermediates and crosslink them. It is one of the surprising featuresof the invention that satisfactory stability of the immobilised arraymay be achieved in the absence of such crosslinking agents or PRPcrosslinking steps. The absence of a polyunsaturated crosslinking agent(such as bisacrylamide or pentaerythritol tetraacrylate) is a preferredfeature in all aspects of this invention directed toward hydrogels, oruses or methods of the preparation thereof. Thus it is a preferredfeature of such aspects of the invention that the mixture to bepolymerised does not comprise such a polyunsaturated crosslinking agentand that the monomers to be polymerised consist essentially of thosedefined in claim 1 (i.e. no polyunsaturated crosslinking monomer isincluded in the mixture).

Generally, in producing hydrogels according to this invention, only onecompound of formulae (I) or (II) will be used.

We have found that use of a compound of the formulae (I) or (II) permitsformation of a hydrogel capable of being immobilised to solid supports,preferably silica-based solid supports. The compounds of these formulaecomprise portions A, B and C as defined herein.

Biradical A. may be oxygen or N(R) wherein R is hydrogen or a C₁₋₅ alkylgroup. Preferably, R is hydrogen or methyl, particularly hydrogen. WhereR is a C₁₋₅ alkyl group, this may contain one or more, e.g. one to threesubstituents. Preferably, however, the alkyl group is unsubstituted.

Biradical B is a predominantly hydrophobic linking moiety, connecting Ato C and may be an alkylene biradical of formula —(CH₂)_(n)—, wherein nis from 1 to 50. Preferably n is 2 or more, e.g. 3 or more. Preferably nis 2 to 25, particularly 2 to 15, more particularly 4 to 12, for example5 to 10.

Where n in —(CH₂)_(n)— is 2 or more, one or more biradicals—CH₂CH₂-(-ethylene-) may be replaced with ethenylene or ethynylenebiradicals. Preferably, however, the biradical B does not contain suchunsaturation.

Additionally, or alternatively, where n in —(CH₂)_(n)— is 1 or more, oneor more methylene radicals —CH₂— in B may be replaced with a mono- orpolycyclic biradical which preferably comprises 5 to 10 carbon atomse.g. 5 or 6 carbon atoms. Such cyclic biradicals may be, for example,1,4-, 1,3- or 1,2-cyclohexyl biradicals. Bicyclic radicals such asnaphthyl or decahydronaphthyl may also be employed. Correspondingheteroatom-substituted cyclic biradicals to those homocyclic biradicalsmay also be employed, for example pyridyl, piperidinyl, quinolinyl anddecahydroquinolinyl.

It will be appreciated that the scope of —B— is thus not particularlyrestricted. Most preferably, however, —B— is a simple, unsubstituted,unsaturated alkylene biradical such as a C₃-C₁₀ alkylene group,optimally C₅-C₈, such as n-pentylene: —(CH₂)₅—.

Where an alkyl group (or alkylene, alkenylene etc) is indicated as being(optionally) substituted, substituents may be selected from the groupcomprising hydroxyl, halo (i.e. bromo, chloro, fluoro or iodo),carboxyl, aldehyde, amine and the like. The biradical —B— is preferablyunsubstituted or substituted by fewer than 10, preferably fewer than 5,e.g. by 1, 2 or 3 such substituents.

Group C serves to permit attachment of molecules of interest afterformation of the hydrogel. The nature of Group C is thus essentiallyunlimited provided that it contains a functionality allowing reactionbetween the hydrogel and molecules of interest. Preferably, such afunctionality will not require modification prior to reaction with themolecule of interest and thus the C group is ready for direct reactionupon formation of the hydrogel.

Preferably such a functionality is a hydroxyl, thiol, amine, acid (e.g.carboxylic acid), ester and haloacetamido, haloacetamido and inparticular bromoacetamido being particularly preferred. Otherappropriate C groups will be evident to those skilled in the art andinclude groups comprising a single carbon-carbon double bond which iseither terminal (i.e. where a C group has an extremity terminating in acarbon-carbon double bond) or where the carbon-carbon double bond is notat a terminal extremity. When a C group comprises a carbon-carbon doublebond, this is preferably fully substituted with C₁₋₅ alkyl groups,preferably methyl or ethyl groups, so that neither carbon atom of theC═C moiety bears a hydrogen atom.

The C moiety may thus comprise, for example, a dimethylmaleimide moietyas disclosed in U.S. Pat. No. 6,372,813, WO01/01143, WO02/12566 andWO03/014394.

The (meth)acrylamide or (meth)acrylate of formula (I) or (II) which iscopolymerised with acrylamide, methacrylamide, hydroxyethyl methacrylateor N-vinyl pyrrolidinone is preferably an acrylamide or acrylate, i.e.,of formula (I). More preferably it is an acrylamide and still morepreferably it is an acrylamide in which A is NH.

The reaction between a comonomer of formula (I) or (II) and acrylamide,methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinonemethacrylamide, particularly acrylamide, has been found to affordhydrogels particularly suitable for use in the generation of moleculararrays. However, it will be appreciated by those skilled in the art thatanalogous copolymers may be formed by the reaction between comonomers offormula (I) or (II) and any vinylogous comonomer,hydroxyethylmethacrylate and n-vinyl pyrrolidinone being two examples ofsuch vinylogous comonomers.

Control of the proportion of monomer of formula (I) or (II) to that ofthe first comonomer (e.g. acrylamide and/or methacrylamide, preferablyacrylamide) allows adjustment of the physical properties of the hydrogelobtained on polymerisation. It is preferred that the comonomer offormula (I) or (II) is present in an amount of mol %, preferably mol %(relative to the total molar quantity of comonomers) in order for thehydrogel to have optimum thermal and chemical stability under conditionstypically encountered during the preparation, and subsequentmanipulation, of the molecular arrays produced from the hydrogels.Preferably, the amount of comonomer of formula (I) or (II) is less thanor equal to about 5 mol %, more preferably less than or equal to about 4mol %. Typical amounts of comonomer of formula (I) or (II) used are1.5-3.5 mol %, exemplified herein by about 2% and about 3%.

The amounts of acrylamide or methacrylamide from which the hydrogels areprimarily constructed are those typically used to form hydrogels, e.g.about 1 to about 10% w/v, preferably less than 5 or 6% w/v, e.g. about 1to about 2% w/v. Again, of course, the precise nature of the hydrogelmay be adjusted by, in part, control of the amount of acrylamide ormethacrylamide used.

When forming the hydrogels, acrylamide or methacrylamide may bedissolved in water and mixed with a solution of a comonomer of formula(I) or (II). The latter may be conveniently dissolved in awater-miscible solvent, such as dimethylformamide (DMF), or wateritself. The most appropriate solvent may be selected by the skilledperson and shall depend upon the structure of the comonomer of formula(I) or (II).

The methods by which the monomers of formula (I) or (II) are synthesisedwill be evident to those skilled in the art. By way of example, thesynthesis of a particularly preferred monomer (of formula (I) wherein ANH, —B—═—(CH₂)₅— and —C═—N(H)—C(═O)CH₂Br is provided as an examplehereinafter.

As noted above, the general methods by which the polymerisation iscarried out are known to those skilled in the art. For example,generally acrylamide or methacrylamide is dissolved in purified water(e.g. MilliQ) and potassium or ammonium persulfate dissolved separatelyin purified water. The comonomer of formula (I) or (II) may beconveniently dissolved in a water-miscible organic solvent, e.g.glycerol, ethanol, methanol, dimethylformamide (DMF) etc. TEMED may beadded as appropriate. Once formulated (a typical preparation isdescribed in the examples), the mixture is polymerised with as littledelay as possible after its formulation.

The polymerisation process may be conducted by any convenient means.Several examples are described in the experimental section below.

The hydrogels according to this invention are of particular utility inthe preparation of arrays of molecules, particularly single moleculearrays (SMAs) or clustered arrays and in particular SMAs or clusteredarrays of polynucleotides.

It is noted above that it is a surprising feature of relevant aspectsthis invention that it is possible to omit the inclusion of apolyunsaturated crosslinking agent. Where such a crosslinking agent isomitted, as is preferable, it is possible to make thinner hydrogels thanhave been achievable heretofore. In particular, omission of suchcrosslinking agents allows preparation of hydrogels having thicknessesof less than about 100 nm, for example less than 75 nm; the hydrogelsmay be less than about 50 nm thick.

Such hydrogels are of particular use where they are used to generatearrays, in particular single molecule arrays or clustered arrays,particularly of nucleotides, and in the interrogation of such arrayswherein fluorescently labelled nucleotides are incorporated into anascent polynucleotide and then detected. Such techniques are describedin greater detail hereinafter.

There are a number of advantages of the hydrogels produced according tothis invention. With particular regard to silica-based substrates, theinvention allows the avoidance of the covalent chemical modification(especially with silicon-containing agents) of the silica-basedsubstrate in order for the hydrogels produced on the support beimmobilised thereto. Thus, the invention permits immobilisation ofhydrogels on a variety of solid supports upon which such immobilisationhas not been reported previously.

By “immobilising” of a hydrogel on a support is meant that the supportedhydrogels are associated with the support in such a way so as to remainas a layer upon the support under conditions encountered duringpreparing and manipulating (e.g. interrogating) molecular arrays. Suchpreparations and manipulations are described in greater detailhereinafter and are known to those skilled in the art.

A further advantage of the supported hydrogels of the invention isavoidance of the need for chemical modification of the hydrogel (i.e.post polymerisation) in order to attach of molecules of interest. Withthe “e” groups described herein, appropriately functionalised moleculesmay be attached directly to the hydrogel.

A further advantage is the passivity, i.e. the essential lack ofreactivity, of the surface of the hydrogel towards non-specificadherence of molecules (e.g. fluorescently labelled nucleotides) to thesurface; such adherents would otherwise create unwanted “noise” duringsubsequent manipulation of molecular arrays formed from the hydrogels,particularly in the manipulation of SMAs or clustered arrays.

As noted above, it has been found that omission of a covalentsurface-modification step results in a surface having greater passivitythan in the prior art, particularly when compared to those instanceswhere the use of the silane-modifying agents described above withsilica-based substrates are employed.

The provision of a surface which leads to as little as possibleaspecific surface contamination is clearly advantageous where thehydrogels are used to construct arrays to be used in SMA applications.Of course, where the hydrogels are used to construct clusteredmicroarrays, minimising surface contamination to the greatest extentpossible is also advantageous.

In accordance with preferred aspects of the invention, thesolid-supported hydrogel-based molecular arrays of the invention may betreated with polyelectrolyte or neutral polymers to afford arrays,preferably SMAs or clustered arrays, particularly SMAs or clusteredarrays of polynucleotides, the surfaces of which have enhanced passivitytowards, for example, aspecific binding with nucleotides (e.g. labellednucleotides) used in sequencing and other interrogative methods of usingthe arrays. Also, the invention, as described and claimed herein,provides an improved method by which molecules of interest (preferablybiomolecules such as those identified above), preferably polynucleotidesand proteins (especially preferably polynucleotides) may be displayed bymodifying existing, i.e. preprepared, arrays of molecules. It will beappreciated that, since this aspect of the invention lies in themodification of existing molecular arrays, the nature of the moleculararray treated according to the method of the invention is not of anyparticular importance. This notwithstanding, it is preferred that thearrays treated are in accordance with the fourth aspect of thisinvention. Likewise, the nature of the biomolecules arrayed, or themeans by which they are arrayed, is of lesser importance than therequirement for the modification step whereby the array is treated withpolyelectrolyte or neutral polymers.

On account of the particular utility of molecular arrays, preferablySMAs or clustered arrays, most preferably SMAs or clustered arrays ofpolynucleotides, in sequence determination methods, the precedingdiscussion has focused, and subsequent discussion shall focus, on thisutility of the invention although it is to be understood that theinvention is not to be considered to be so limited.

Similarly, the make-up of the solid support of the array to be modified,or the type of the array to be modified is not of as great importance asthe manner in which it is treated (modified) according to the invention.SMAs and clustered arrays, however, and preferably SMAs and clusteredarrays of polynucleotides are particularly advantageous.

The term “single molecule array” or “SMA” as used herein refers to apopulation of polynucleotide molecules, distributed (or arrayed) over asolid support, wherein the spacing of any individual polynucleotide fromall others of the population is such that it is possible to effectindividual resolution, or interrogation, of the polynucleotides. Thetarget nucleic acid molecules immobilised onto the surface of the solidsupport should thus be capable of being resolved by optical means. Thismeans that, within the resolvable area of the particular imaging deviceused, there must be one or more distinct signals, each representing onepolynucleotide. This may be achieved, preferably wherein the spacingbetween adjacent polynucleotide molecules on the array is at least 100nm, more preferably at least 250 nm, still more preferably at least 300nm, even more preferably at least 350 nm. Thus, each molecule isindividually resolvable and detectable as a single molecule fluorescentpoint, and fluorescence from said single molecule fluorescent point alsoexhibits single step photobleaching.

Clusters of substantially identical molecules do not exhibit singlepoint photobleaching under standard operating conditions used todetect/analyze molecules on arrays. The intensity of a single moleculefluorescence spot is constant for an anticipated period of time afterwhich it disappears in a single step. In contrast, the intensity of a‘fluorescence spot comprised of two or more molecules, for example,disappears in two or more distinct and observable steps, as appropriate.The intensity of a fluorescence spot arising from a cluster consistingof thousands' of similar molecules, such as those present on the arraysconsisting of thousands of similar molecules at any given point, forexample, would disappear in a pattern consistent with an exponentialdecay. The exponential decay pattern reflects the progressive loss offluorescence by molecules present in the cluster and reveals that, overtime, fewer and fewer molecules in the spot retain their fluorescence.

The term “clustered array” refers to an array wherein distinct regionsor sites on the array comprise multiple polynucleotide molecules thatare not individually resolvable by optical means. Depending on how thearray is formed each region or site on the array may comprise multiplecopies of one individual polynucleotide molecule or even multiple copiesof a small number of different polynucleotide molecules (e.g. multiplecopies of two complementary nucleic acid strands). In a preferredembodiment the term “clustered array” refers to an array produced bysolid-phase amplification of a target or template polynucleotide,wherein amplified copies of the target or template become covalentlybound to the solid support during amplification.

Clustered arrays of nucleic acid molecules may be produced usingtechniques generally known in the art. By way of example, WO 98/44151and WO 00/18957 both describe methods of nucleic acid amplificationwhich allow amplification products to be immobilised on a solid supportin order to form arrays comprised of clusters or “colonies” ofimmobilised nucleic acid molecules. A further method for the preparationof clustered arrays on a solid-support bound hydrogel is described infurther detail below and in the accompanying examples. It will beappreciated that the arrays of the invention, in all aspects, may bearrays of clusters of molecules. Such arrays are a preferred embodimentof all aspects of the invention.

The support for the molecular array (hydrogel-based or otherwise) whichmay be modified according to the invention is not limited to aparticular matrix or substrate. Supports which may be of use in thepractise of this invention are as described above.

Some supports to which biomolecules, such as polynucleotides, areattached are silica-based supports themselves. In certain embodiments ofthe invention these may be covalently modified in some way so as toallow covalent attachment of either polynucleotides, or to immobilise achemically reactive group hydrogel or a partially formed hydrogel (e.g.a prepolymer). The surface-activating agent is typically anorganosilicon (organosilane) compound such as those listed above.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in WO 97/04131,wherein hairpin polynucleotides are immobilised on a glass support byreaction between a pendant epoxide group on the glass with an internalamino group held within the loop.

Zhao et al. (Nucleic Acids Research, 2001, 29(4), 955959) disclose theformation of a hairpin polynucleotide which contains multiplephosphorothioate moieties in the loop. The moieties are used to anchor,in more than one position, the hairpin DNA to glass slides pre-activatedwith bromoacetamidopropylsilane.

The work of Zhao developed upon earlier work of Pirrung et al.(Langmuir, 2000, 16, 2185-2191) in which the authors report that5′-thiophosphate-terminating oligonucleotides could be attached toglass, pre-activated with mono- and dialkoxylated silanes andbromoacetamide.

In addition, we disclose in our copending International patentapplication number PCT/GB2004/004707 arrays of hairpin polynucleotidesattached to a solid support, e.g. for use in the preparation of SMAs, byreaction of a sulfur-based nucleophile with the solid support. Thesulfur-based nucleophile may be directly attached to the hairpinalthough it is preferably indirectly attached through a linker.Attachment is by way of an internal nucleotide within the hairpin, thatis to say that the sulfur-based nucleophile is not connected directly orthrough a linker to a nucleotide at either terminus of the hairpin.

Still further example of arrays are those in which biomolecules,preferably polynucleotides, are attached to hydrogels supported uponsilica-based or other solid supports. Silica-based supports aretypically used to support hydrogels and hydrogel arrays as described inWO00/31148, WO01/01143, WO02/12566, WO03/014392, U.S. Pat. No. 6,465,178and WO00/53812.

The solid supports for such hydrogels are preferably silica-based sincethe silica-based support need not be covalently modified bypreactivation with a silylating agent as described above in order toimmobilise the hydrogel thereto. Clearly, however, such hydrogels maystill be attached to silica-based supports which have beensurface-activated, e.g. with an organosilane molecule as describedabove.

A further type of molecular array which may be treated according to thisinvention are PEM-supported molecular arrays of the type described byBraslaysky et al. (infra) I and Kartlov et al. (infra).

There are thus three main types of molecular array which may be treatedaccording to this invention:

(1) arrays directly supported onto silica-based supports;

(2) hydrogel-based molecular arrays; and

(3) PEM-supported molecular arrays.

Of these the hydrogel-based molecular arrays are most preferred, and inparticular hydrogel-based arrays according to the fourth aspect of theinvention primarily because of the simplicity with which these may beconstructed. Whilst, such hydrogels are advantageous on account of thepassivity of the surface, we have found that the surface treatment ofthis invention leads to still greater passivity, and thus utility insequencing reactions and the like, of the resultant molecular arrays.

According to this invention, the surface of an existing array ofbiomolecules, preferably of polynucleotides, is modified by treatmentwith a mixture comprising polyelectrolyte a mixture comprising neutralpolymers, or a mixture comprising both polyelectrolytes and neutralpolymers.

Polyelectrolytes are large, generally polymeric, molecules containing aplurality of ionisable groups. Examples include polyallylamine (PAL),commercially available as polyallylamine hydrochloride (PAL.HCl),polyacrylic acid (PAA), poly(styrene sulfonate) (PSS) andpolyethyleneimine (PEI). The degree to which they are ionised isdependent upon the pH of the medium in which they are present.

In the context of DNA sequencing, we have found that a combination ofmore than one polyelectrolyte is particularly advantageous whenmodifying molecular arrays. As an example, we have found that thesequential application of PAL.HCl followed by PAA to be particularlypreferred.

The conditions under which the arrays may be treated include exposingthem to solutions, or suspensions of polyelectrolyte or neutral polymer.Preferably these solutions or suspensions are aqueous. Particularlypreferably the pH of the solution or suspension is higher than 6 andless than 8.5, more preferably from 6.5 to 8, still more preferably from6.5 to 7.5, more preferably approximately neutral, or about pH 7.

As an alternative to polyelectrolytes, neutral polymers, e.g.polyethylene glycols, such as those commercially available, e.g. PEG8000 available from Sigma, may be used.

Of course both polyelectrolyte and neutral polymers may be used.

It will be appreciated that the treatment of molecular arrays accordingto this invention shall generally, particularly in the case of planararrays, serve to deposit layers of polyelectrolyte and/or polymers.

When polynucleotides are arrayed in molecular arrays according to thevarious aspects of this invention, including the solid-supportedhydrogel-based molecular arrays of the invention, these are preferablyhairpin polynucleotides comprising a polynucleotide duplex which may beused to retain a primer and a target polynucleotide in spatialrelationship. Preferably the target polynucleotide is present at the 5′end and the primer is present at the 3′ end although hairpinpolynucleotides where the primer is present at the 5′ end and the targetpolynucleotide is present at the 3′ end are also embraced by thisinvention.

As used herein, the term “interrogate” can refer to any interaction of amolecule on the array with any other chemical or molecule and may alsorefer to any analysis of a detectable signal from a molecule on thearray or any other molecule which is bound thereto or associatedtherewith. In one embodiment “interrogation” encompasses a targetpolynucleotide on the array functioning as a template upon which DNApolymerase acts. In other words, “interrogating” can encompasscontacting the target polynucleotides with another molecule, e.g., apolymerase, a nucleoside triphosphate, a complementary nucleic acidsequence, wherein the physical interaction provides informationregarding a characteristic of the arrayed target polynucleotide. Thecontacting can involve covalent or non-covalent interactions with theother molecule. As used herein, “information regarding a characteristic”means information about the identity or sequence of one or morenucleotides in the target polynucleotide, the length of thepolynucleotide, the base composition of the polynucleotide, the Tm ofthe polynucleotide, the presence of a specific binding site for apolypeptide, a complementary nucleic acid or other molecule, thepresence of an adduct or modified nucleotide, or the three-dimensionalstructure of the polynucleotide.

The spatial relationship between primer and target polynucleotidepresent in hairpin polynucleotides permits improved sequence analysisprocedures to be conducted. Maintenance of the spatial relationship ismade possible not only by the hydrogen bonds formed on hybridisation,but also by the tethering of a known primer to the targetpolynucleotide. The fixing of the primer, as part of the hairpinstructure, to the hydrogel support, ensures that the primer is able toperform its priming function during a polymerase-based sequencingprocedure, and is not removed during any washing step in the procedure.

There are many different ways of forming a hairpin structure so as toincorporate the target polynucleotide. A preferred method is to form afirst molecule (which may contain a non-backbone sulfur-basednucleophile attached through a linker) capable of forming a hairpinstructure, and ligate the target polynucleotide to this. It is possibleto ligate any desired target polynucleotide to the hairpin constructbefore or after arraying the hairpins on the solid support.Alternatively, a first polynucleotide may be ligated before arraying anda second ligated after arraying. It is, of course, also possible tointroduce a nucleophile (preferably a sulfur-based nucleophile) aftersuch a ligation.

Where a target polynucleotide is a double-stranded DNA, this may beattached to the stem of the hairpin by ligating one strand to thehairpin polynucleotide and removing the other strand after the ligation.

The target polynucleotide may be genomic DNA purified using conventionalmethods. The genomic DNA may be PCR-amplified or used directly togenerate fragments of DNA using either restriction endonucleases, othersuitable enzymes, a mechanical form of fragmentation or a non-enzymaticchemical fragmentation method. In the case of fragments generated byrestriction endonucleases, hairpin structures bearing a complementaryrestriction site at the end of the first hairpin may be used, andselective ligation of one strand of the DNA sample fragments may beachieved by one of two methods.

Method 1 uses a hairpin containing a phosphorylated 5′ end. Using thismethod, it may be necessary to first de-phosphorylate therestriction-cleaved genomic or other DNA fragments prior to ligationsuch that only one sample strand is covalently ligated to the hairpin.

Method 2: in the design of the hairpin, a single (or more) base gap canbe incorporated at the 3′ end (the receded strand) such that uponligation of the DNA fragments only one strand is covalently joined tothe hairpin. The base gap can be formed by hybridising a furtherseparate polynucleotide to the 5′-end of the first hairpin structure. Onligation, the DNA fragment has one strand joined to the 5′-end of thefirst hairpin, and the other strand joined to the 3′-end of the furtherpolynucleotide. The further polynucleotide (and the other stand of thefragment) may then be removed by disrupting hybridisation.

In either case, the net result should be covalent ligation of only onestrand of a DNA fragment of genomic or other DNA to the hairpin. Suchligation reactions may be carried out in solution at optimisedconcentrations based on conventional ligation chemistry, for example,carried out by DNA ligases or non-enzymatic chemical ligation. Shouldthe fragmented DNA be generated by random shearing of genomic DNA orpolymerase, then the ends can be filled in with Klenow fragment togenerate blunt-ended fragments which may be blunt-end-ligated ontoblunt-ended hairpins. Alternatively, the blunt-ended DNA fragments maybe ligated to polynucleotide adapters which are designed to allowcompatible ligation with the sticky-end hairpins, in the mannerdescribed hereinbefore.

Polynucleotides, particularly hairpin polynucleotides, may be bounddirectly to the “C” groups of hydrogels, if present, by, for example,immobilising them through a covalent bond between each polynucleotide(by way of a nucleophile, preferably sulfur-based nucleophile) and the“C” group. In doing so it is thus possible to generate arrays, e.g.microarrays or SMAs, preferably SMAs, of the hairpin polynucleotides.

The precise density of the arrays is not critical. For single moleculeresolution, in fact, the higher the density of hairpin polynucleotidemolecules arrayed the better since more information may be obtained fromanyone experiment. For example, there may be at least 10³ molecules/cm²,preferably at least 10⁵ molecules/cm′ and most preferably 10⁶-10⁹molecules/cm². Particularly preferably, the density of sample moleculesis at least 10⁷/cm², typically it is approximately 10⁸-10⁹/cm².

Such “high density” arrays are in contrast to those arrays such as thoseso described in the prior art which are not necessarily as high or, e.g.in the many molecule arrays of—Fodor et al (supra), which compriseclusters of polynucleotides comprising a plurality of tightly packedpolynucleotides that are resolvable at the level of the cluster, not atthe level of the polynucleotides, are too high to allow single moleculeresolution. By arraying the polynucleotides at a density that they canbe considered to be single molecules, i.e. each can be individuallyresolved, a SMA is created.

The terms “individually resolved” and “individual resolution” are usedherein to specify that, when visualised, it is possible to distinguishone molecule on the array from its neighbouring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules. Itwill usually be the target polynucleotide portion that is individuallyresolved, as it is this component which is intended to be interrogated,e.g. by the incorporation of detectable bases.

Covalent bonding, where present, between the “C” groups in the hydrogeland polynucleotides (e.g. hairpin polynucleotides or oligonucleotideprimers used for formation of clustered arrays by solid-phaseamplification), may be effected by any convenient means.

Preferably, where the “C” group is a haloacetamido group,polynucleotides bearing sulfur-containing nucleophilic groups are used.Examples of appropriate sulfur nucleophile-containing polynucleotidesare disclosed in Zhao et al (Nucleic Acids Research, 2001, 29(4),955-959) and Pirrung et al (Langmuir, 2000, 16, 2185-2191).

However, the preferred class of sulfur-based nucleophiles pendant fromthe polynucleotides for reaction with the reactive groups on thehydrogels are not particularly restricted. The sulfur-based nucleophilemay thus be a simple thiol (—SH wherein — denotes the bond or linkerconnecting the thiol to the remainder of the polynucleotide). Furtherexamples of sulfur-based nucleophiles include a moiety of the formula(III):

(wherein — denotes the bond or linker connecting the sulfur-basednucleophile to the remainder of the polynucleotide; X represents anoxygen atom, a sulfur atom or a group NR, in which R is hydrogen or anoptionally substituted C1-10 alkyl; Y represents an oxygen or a sulfuratom; and Z represents an oxygen atom, a sulfur atom or an optionallysubstituted C1-10 alkyl group).

Preferred moieties of formula (III) are those in which X is oxygen orsulfur, preferably oxygen. Where X is a group NR, R is preferablyhydrogen. Y is preferably oxygen. Z is preferably an oxygen or sulfuratom or a methyl group, particularly preferably an oxygen atom.

The preferred sulfur-based nucleophile is thiophosphate although othersulfur-based nucleophiles described are also of utility, for examplethiophosphoramidates.

Particularly preferably, the sulfur-containing nucleophiles describedherein are connected to the polynucleotide through a linker group. Thelinker may be a carbon-containing chain such as those of formula (CH₂)nwherein “n” is from 1 to about 1500, for example less than about 1000,preferably less than 100, e.g. from 2-50, particularly 5-25. However, avariety of other linkers may be employed with the only restrictionplaced on their structures being that the linkers are stable underconditions under which the polynucleotides are intended to be usedsubsequently, e.g. conditions used in DNA sequencing.

Linkers which do not consist of only carbon atoms may also be used. Suchlinkers include polyethylene glycol (PEG) having a general formula of(CH₂—CH₂—O)_(m), wherein m is from about 1 to 600, preferably less thanabout 500.

Linkers formed primarily from chains of carbon atoms and from PEG may bemodified so as to contain functional groups which interrupt the chains.Examples of such groups include ketones, esters, amines, amides, ethers,thioethers, sulfoxides, sulfones. Separately or in combination with thepresence of such functional groups may be employed alkene, alkyne,aromatic or heteroaromatic moieties, or cyclic aliphatic moieties (e.g.cyclohexyl). Cyclohexyl or phenyl rings may, for example, be connectedto a PEG or (CH₂)_(n) chain through their 1- and 4-positions.

Examples of appropriately modified linkers are those of formula(CH₂)_(n) (wherein n is as defined above) and in which one or more CH₂units are replaced with functional groups). Thus, one or more CH₂ unitsmay be exchanged for an oxygen to form an ether, or for a SO₂ to form asulfone etc. One or more CH₂ units may be exchanged for an amide moietyor alkene or alkyne unit. In such linkers one or more functional groupsmay be present; these functional groups may or may not be the same aseach other. Linkers of particular interest contain the propargylaminounit attached to the base (e.g. uracil) in a modified nucleotide. Suchnucleotides contain the following unit:

The amino group may be connected to the remainder of the linker byformation of an amide bond.

Modified nucleotides are commercially available, e.g. from the DNAsynthesis company Oswell (now Eurogentec Group). Such nucleotidesinclude 3′OH capped nucleotides which may be a basic where a cappedlinker is attached at the 1′ carbon atom or contain a base to which acapped linker is attached. Two such modified nucleotides are Oswellproducts OSW428 and OSW421:

Those skilled in the art will be aware of methods of deprotecting thefluorenylmethoxycarbonyl (Fmoc) group which caps the linker in thenucleotides shown above and for effecting terminal modification, e.g.thiophosphorylation, of the linker.

As an alternative to the linkers described above, which are primarilybased on linear chains of saturated carbon atoms, optionally interruptedwith unsaturated carbon atoms or heteroatoms, other linkers may beenvisaged which are based on nucleic acids or monosaccharide units (e.g.dextrose). It is also within the scope of this invention to utilisepeptides as linkers.

Longer linker moieties (e.g. those containing a chain or more than 100atoms, particularly those in excess of 500 or even 1000 atoms) serve toposition the polynucleotide further away from the solid support. Thisplaces the polynucleotide (e.g. DNA) in a environment having a greaterresemblance to free solution. This can be beneficial, for example, inenzyme-mediated reactions effected on the polynucleotide. This isbecause such reactions suffer less from the steric hindrance whichmanifests itself where the polynucleotide is directly attached to thesupport or is indirectly attached through a very short linker (such as alinker comprising a chain or only several, e.g. about 1 to 3 carbonatoms).

Where the molecules of interest immobilised on the array arepolynucleotides the “linker” may comprise one or more nucleotides whichform part of the polynucleotide but which do not participate in anyreaction carried out on or with the polynucleotide (e.g. a hybridisationor amplification reaction). Such nucleotides may be referred to hereinas “spacer” polynucleotides. Typically from 1 to 20, more preferablyfrom 1 to 15 or from 1 to 10, and more particularly 2, 3, 4, 5, 6, 7, 8,9 or 10 spacer nucleotides may be included. Most preferably thepolynucleotide will include 10 spacer nucleotides. It is preferred touse polyT spacers, although other nucleotides and combinations thereofcan be used. In the most preferred embodiment the polynucleotide willinclude 10T spacer nucleotides.

Spacer nucleotides are typically included at the 5′ ends ofpolynucleotides which are attached to a suitable support, for example asolid-supported hydrogel, via a linkage with the 5′ end of thepolynucleotide. Attachment can be achieved through a sulphur-containingnucleophile, such as phosphorothioate, present at the 5′ end of thepolynucleotide. In the case of arrays based on solid-supportedhydrogels, this nucleophile is bound to a “e” group present in thehydrogel. The one or more spacer nucleotides function to space theportions of the polynucleotide that will be “interrogated” and/orsubject to further manipulations, such as hybridisation to a secondpolynucleotide, away from the site of attachment to the solid support.The present inventors have observed that inclusion of spacer nucleotidesat the 5′ end can markedly improve the performance of hybridisation ofcomplementary polynucleotides to target regions of the immobilisedpolynucleotides downstream (3′) of the spacer nucleotides. Hybridisationyield is observed to increase sharply with polyT spacers of from 2 to10T nucleotides. When spacer length is increased from 10T up to 20Thybridisation yield begins to decrease. In the most preferred embodimentthe polynucleotide will include 10T spacer nucleotides and a 5′phosphorothioate group.

Whilst the use of sulfur nucleophile-containing polynucleotides(particularly thiophosphate-containing) and haloacetamide e groups arepreferred to effect attachment of polynucleotides in all aspects of theinvention, the skilled person will be able to envisage many othercombinations of functionality which will facilitate immobilisation ofpolynucleotides to the hydrogels described herein. For example, the Cgroup may comprise an activated ester and the polynucleotide may bear anamino group or an oxygen-based nucleophile. Other combinations will beevident to those skilled in the art.

In a particular embodiment the invention provides a method of forming aclustered array on a solid-supported hydrogel by means of a nucleic acidamplification reaction.

Therefore, in a further aspect the invention provides a method ofpreparing a solid supported hydrogel-based molecular array which is aclustered array of molecules of interest, the method comprising:

-   -   (i) reacting polynucleotide molecules with reactive sites        present in a solid-supported hydrogel, wherein said        polynucleotide molecules are first and second oligonucleotide        primers capable of hybridising to a template to be amplified;    -   (ii) contacting the first oligonucleotide primers attached to        the solid-supported hydrogel in step (i) with one or more        templates to be amplified under conditions which permit        hybridisation of the templates to the oligonucleotide primers,        each template comprising at the 3′ end a sequence capable of        hybridising to the first oligonucleotide primer and at the 5′        end a sequence the complement of which is capable of hybridising        to a second oligonucleotide primer; and    -   (iii) performing one or more nucleic acid amplification        reactions using the first and second oligonucleotide primers and        the template(s), thereby generating a clustered array of        molecules of interest.

In this method a solid-supported hydrogel is provided, preferablyprepared using the method according to the first aspect of theinvention. Oligonucleotide primers are then linked or “grafted” to thehydrogel via reaction with reactive sites present on the hydrogel. Thisstep can be carried out as described above and all preferred featuresdescribed apply mutatis mutandis to this aspect of the invention. Theoligonucleotide primers are preferably attached to the hydrogel viacovalent linkage their 5′ ends leaving the 3′ end of the molecule freeto participate in hybridisation to a template polynucleotide andsubsequent primer extension by addition of further nucleotides to thefree 3′ end of the primer. Attachment could also be effected via aninternal nucleotide in the primer, provided that this does not preventthe primer from hybridising to a template and subsequent primerextension. The most preferred means of attachment is via 5′phosphorothioate to a hydrogel comprised of polymerised acrylamide andBRAPA.

The precise sequence of the primer oligonucleotides will be dependent onthe nature of the template it is intended to amplify. The first andsecond primers may be of different or identical sequence. The primerscan include natural and non-natural bases or any combination thereof,and may also include non-natural backbone linkages such asphosphorothioate. The primer may advantageously' include spacernucleotides, as described above, in order to optimise the efficiency ofsubsequent hybridisation to the template polynucleotide. The primer maycontain from 1 to 20, more preferably from 1 to 15 or from 1 to 10, andmore particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 spacer nucleotides. Mostpreferably the primer will include 10 spacer nucleotides. It ispreferred to use polyT spacers, although other nucleotides andcombinations thereof can be used. In the most preferred embodiment theprimer will include 10T spacer nucleotides.

The primer oligonucleotides are grafted onto the surface of thesolid-supported hydrogel, effectively forming a surface that is ready tobe used for nucleic acid amplification. This approach contrasts withprior art methods for amplification on solid supports, such as thatdescribed in WO 98/44151 (schematically illustrated in FIG. 5), whereina mixture of primers and templates are grafted to the solid surfacesimultaneously in a single grafting step. In this approach a specificgrafting mixture of primers and templates has to be used for eachspecific template to be amplified. In addition, grafting of longtemplate nucleic acid fragments (>300 bp) is technically difficult. Theinventors' approach avoids this problem by removing the need to graftprimers and templates simultaneously. In the method of the inventionprimers are grafted in the absence of template to form a surface that isready for hybridisation to the template and subsequent amplification.

Following attachment of the primers the solid support is contacted withthe template to be amplified under conditions which permit hybridisationbetween the template and the bound primers. The template is generallyadded in free solution and suitable hybridisation conditions will beapparent to the skilled reader. Typically hybridisation conditions are5×SSC at 40° C., following an initial denaturation step.

The template polynucleotide (or a denatured single strand thereof ifreferring to a template duplex) will include at the 3′ end a sequencewhich permits hybridisation to a first oligonucleotide primer and at the5′ end of the same strand a sequence, the complement of which permitshybridisation to a second oligonucleotide primer (i.e. the sequence ofthe second primer may be substantially identical to the sequence at the5′ end of the template). The remainder of the template can be anypolynucleotide molecule that it is desired to amplify to form aclustered array. Templates may be, for example, fragments of genomic DNAor cDNA that it is desired to sequence. The sequences permittinghybridisation to primers will typically be around 20-25 nucleotides inlength. The term “hybridization” encompasses sequence-specific bindingbetween primer and template. Binding of the primer to its cognatesequence in the template can occur under typical conditions used forprimer-template annealing in standard PCR.

Following hybridisation of the templates to primers bound to the solidsupport a nucleic acid amplification reaction can be carried out usingthe bound primers and the hybridised template. The first step of theamplification reaction will be a primer extension step, in whichnucleotides are added to the free 3′ ends of the bound primers in orderto synthesise complementary strands corresponding to the full length ofthe template (illustrated schematically in FIG. 6). Subsequentdenaturation results in a full-length complementary template strandcovalently bound to the solid support. This complementary strand willthus include at its 3′ end a sequence which is capable of binding to thesecond oligonucleotide primer. Further rounds of amplification(analogous to a standard PCR reaction) lead to the formation of clustersor colonies of template molecules bound to the solid support.

DNA amplification on solid supports is a procedure well documented inthe literature. A wide range of support types (e.g. microarrays (HuberM. et al. (2001) Anal. Biochem. 299(1), 24-30; Rovera G. (2001) U.S.Pat. No. 6,221,635 B1 20010424), glass beads (Adessi C. et al. (2000)Nucl. Acids Res. 28(20) I e87; Andreadis J. D. et al. (2000) Nucl. AcidsRes. 28(2), ed), agarose (Stamm S. et al. (1991) Nucl. Acids Res. 19(6),1350) or polyacrylamide (Shapero M. H. et al. (2001) Genome Res. 11,1926-1934; Mitra, R. D. et al. (1999) Nucl. Acids Res. 27(24), e34)) andattachment chemistries (e.g. 5′-thiol oligo on aminosilane slides viaheterofunctional crosslinker (Adessi C. et al. (2000) Nucl. Acids Res.28(20), e87; Andreadis J. D. et al. (2000) Nucl. Acids Res. 28(2), e5),EDC chemistry on NucleoLink™ surface (Sjoroos M. et al. (2001) Clin.Chem. 47 (3), 498-504) or amino silane (Adessi C. et al. (2000) Nucl.Acids Res. 28(20), e87), radical polymerization (Shapero M. H. et al.(2001) Genome Res. 11, 1926-1934; Mitra, R. D. et al. (1999) Nucl. AcidsRes. 27(24), e34)) have been described. PCR on polyacrylamide coatedglass slides (Shapero et al., ibid) or beads (Mitra et al., ibid) hasalso been reported. In both cases, at least one of the primers containsa 5′-acrylamide modification so that the primer is covalently attachedto the solid support through copolymerization. The method of Mitra etal. consists in premixing of all reagents necessary to perform the PCR,the primers and a very low concentration of the template, thenpolymerizing a thin polyacrylamide film onto glass slides. In the methodof Shapero et al., the primers are both copolymerized while coating thebeads and the template is introduced by hybridization later on.

One of the major drawbacks of both methods is the lack of versatility ofthe surface because of the introduction of the primers with or withoutthe template during preparation of the surface. Moreover, the primers orthe template can be potentially damaged by the free radicals generatedduring the polyacrylamide polymerization.

WO 98/44151 describes the use of a hybridisation technique of a templateto a primer followed by a chain extension and then replication by PCR togenerate colonies or clusters of immobilised nucleic acids on a solidsupport. The cluster technology as described in WO 98/44151 involves agrafting step that consists of immobilizing simultaneously both primersand templates on a carboxylated surface using standard coupling (EDC)chemistry. This attachment is covalent and has to be done using aspecific reaction mixture for every template used. The strategy used isillustrated in FIG. 5.

The inventors' approach involves an initial step of preparing thesurface of the solid support, followed by covalent attachment (grafting)of primers to generate a surface ready for use in PCR. The PCR templatemay then be hybridised to attached primers immediately prior to the PCRreaction. The PCR reaction thus begins with an initial primer extensionstep rather than template denaturation. This approach is illustrated inFIG. 6.

Uses of Arrays Prepared According to the Invention

Once formed, arrays prepared according to the invention may be used inessentially any method of analysis which requires interrogation ofmolecules of interest on the array or of molecules bound to molecules ofinterest on the array. In this context, molecules “bound” to moleculesof interest on the array includes complementary polynucleotide strandshybridised to polynucleotides bound on the array. By way of example, thearrays may be used to determine the properties or identities of cognatemolecules. Typically, interactions between biological or chemicalmolecules with molecules of interest bound on the arrays are carried outin solution. In preferred embodiments the arrays may be used inprocedures to determine the sequence of polynucleotides on the array,and also in the identification and/or scoring of single nucleotidepolymorphisms, gene expression analysis, etc.

In particular, the arrays may be used in assays which rely on thedetection of fluorescent labels to obtain information on the arrayedmolecules, typically arrayed polynucleotides. The arrays areparticularly suitable for use in multi-step assays. The arrays may beused in conventional techniques for obtaining genetic sequenceinformation. Many of these techniques rely on the stepwiseidentification of suitably labelled nucleotides, referred to in U.S.Pat. No. 5,654,413 as “single base” sequencing methods or“sequencing-by-synthesis”.

In an embodiment of the invention, the sequence of a targetpolynucleotide is determined in a similar manner to that described inU.S. Pat. No. 5,654,413, by detecting the incorporation of one or morenucleotides into a nascent strand complementary to the targetpolynucleotide to be sequenced through the detection of fluorescentlabel(s) attached to the incorporated nucleotide(s). Sequencing of thetarget polynucleotide is primed with a suitable primer (or prepared as ahairpin construct which will contain the primer as part of the hairpin),and the nascent chain is extended in a stepwise manner by addition ofnucleotides to the 3′ end of the primer in a polymerase-catalysedreaction.

In preferred embodiments each of the different nucleotides (A, T, G andC) is labelled with a unique fluorophore which acts as a blocking groupat the 3′ position to prevent uncontrolled polymerisation. Thepolymerase enzyme incorporates a nucleotide into the nascent chaincomplementary to the target polynucleotide, and the blocking groupprevents further incorporation of nucleotides. The array surface is thencleared of unincorporated nucleotides and each incorporated nucleotideis “read” optically by suitable means, such as a charge-coupled deviceusing laser excitation and filters. The 3′-blocking group is thenremoved (deprotected), to expose the nascent chain for furthernucleotide incorporation.

Similarly, U.S. Pat. No. 5,302,509 discloses a method to sequencepolynucleotides immobilised on a solid support. The method relies on theincorporation of fluorescently-labelled, 3′-blocked nucleotides A, G, Cand T into a growing strand complementary to the immobilisedpolynucleotide, in the presence of DNA polymerase. The polymeraseincorporates a base complementary to the target polynucleotide, but isprevented from further addition by the 3′-blocking group. The label ofthe incorporated base can then be determined and the blocking groupremoved by chemical cleavage to allow further polymerisation to occur.

In the case of single molecule arrays, because the array consists ofdistinct optically resolvable polynucleotides, each targetpolynucleotide will generate a series of distinct signals as thefluorescent events are detected. The sequence of the targetpolynucleotide is inferred from the order of addition of nucleotides inthe complementary strand following conventional rules of base-pairing.

The term “individually resolved by optical microscopy” is used herein toindicate that, when visualised, it is possible to distinguish at leastone polynucleotide on the array from its neighbouring polynucleotidesusing optical microscopy methods available in the art. Visualisation maybe effected by the use of reporter labels, e.g., fluorophores, thesignal of which is individually resolved.

Other suitable sequencing procedures will be apparent to the skilledperson. In particular, the sequencing method may rely on the degradationof the arrayed polynucleotides, the degradation products beingcharacterised to determine the sequence.

An example of a suitable degradation technique is disclosed inWO95/20053, whereby bases on a polynucleotide are removed sequentially,a predetermined number at a time, through the use of labelled adaptorsspecific for the bases, and a defined exonuclease cleavage.

A consequence of sequencing using non-destructive methods is that it ispossible to form a spatially addressable array for furthercharacterisation studies, and therefore non-destructive sequencing maybe preferred. In this context, the term “spatially addressable” is usedherein to describe how different molecules may be identified on thebasis of their position on an array.

In the case that the target polynucleotide fragments are generated viarestriction digest of genomic DNA, the recognition sequence of therestriction or other nuclease enzyme will provide 4, 6, 8 bases or moreof known sequence (dependent on the enzyme). Further sequencing ofbetween 10 and 20 bases on the array should provide sufficient overallsequence information to place that stretch of DNA into unique contextwith a total human genome sequence, thus enabling the sequenceinformation to be used for genotyping and more specifically singlenucleotide polymorphism (SNP) scoring.

The sequencing method that is used to characterise the bound target maybe any known in the art that measures the sequential incorporation ofbases onto an extending strand. A suitable technique is disclosed inU.S. Pat. No. 5,302,509 requiring the monitoring of sequentialincorporation of fluorescently-labelled bases onto a complement usingthe polymerase reaction. Alternatives will be apparent to the skilledperson. Suitable reagents, including fluorescently-labelled nucleotideswill be apparent to the skilled person.

Thus the devices into which the arrays of this invention may beincorporated include, for example, a sequencing machine or geneticanalysis machine.

In the case of single molecule arrays the single polynucleotidesimmobilised onto the surface of a solid support should be capable ofbeing resolved by optical means. This means that, within the resolvablearea of the particular imaging device used, there must be one or moredistinct signals, each representing one polynucleotide. Typically, thepolynucleotides of the array are resolved using a single moleculefluorescence microscope equipped with a sensitive detector, e.g., acharge-coupled device (CCD). Each polynucleotide of the array may beimaged simultaneously or, by scanning the array, a fast sequentialanalysis can be performed.

The extent of separation between the individual polynucleotides on thearray will be determined, in part, by the particular technique used toresolve the individual polynucleotide. Apparatus used to image moleculararrays are known to those skilled in the art. For example, a confocalscanning microscope may be used to scan the surface of the array with alaser to image directly a fluorophore incorporated on the individualpolynucleotide by fluorescence. Alternatively, a sensitive 2-D detector,such as a charge-coupled device, can be used to provide a 2-D imagerepresenting the individual polynucleotides on the array.

“Resolving” single polynucleotides on the array with a 2-D detector canbe done if, at 100× magnification, adjacent polynucleotides areseparated by a distance of approximately at least 250 nm, preferably atleast 300 nm and more preferably at least 350 nm. It will be appreciatedthat these distances are dependent on magnification, and that othervalues can be determined accordingly, by one of ordinary skill in theart.

Other techniques such as scanning near-field optical microscopy (SNOM)are available which are capable of greater optical resolution, therebypermitting more dense arrays to be used. For example, using SNOM,adjacent polynucleotides may be separated by a distance of less than 100nm, e.g., 10 nm. For a description of scanning near-field opticalmicroscopy, see Moyer et al., Laser Focus World (1993) 29 (10).

An additional technique that may be used is surface-specific totalinternal reflection fluorescence microscopy (TIRFM) see, for example,Vale et al., Nature (1996) 380:451-453). Using this technique, it ispossible to achieve wide-field imaging (up to 100 μm×100 μm) with singlemolecule sensitivity. This may allow arrays of greater than 10⁷resolvable polynucleotides per cm² to be used.

Additionally, the techniques of scanning tunneling microscopy (Binnig etal., Helvetica Physica Acta (1982) 55:726-735) and atomic forcemicroscopy (Hansma et al., Ann. Rev. Biophys. Biomol. Struct. (1994)23:115-139) are suitable for imaging the arrays of the presentinvention. Other devices which do not rely on microscopy may also beused, provided that they are capable of imaging within discrete areas ona solid support.

Once sequenced, the spatially addressed arrays may be used in a varietyof procedures which require the characterisation of individual moleculesfrom heterogeneous populations.

Polynucleotides bound on clustered arrays may also be used as templatesfor “sequencing-by-synthesis” reactions in which one or more nucleotidesare successively incorporated into growing strands complementary to thetarget polynucleotides to be sequenced and the identity of the base(s}added in one or more of the nucleotide incorporation steps isdetermined. Again sequencing requires a suitable primer complementary tothe template which can serve as an initiation point for the addition offurther nucleotides in the sequencing reaction. The sequence of thebound polynucleotide is inferred from the identity of the incorporatednucleotides following conventional base-pairing rules. Methods fornucleic acid sequencing on clustered arrays or nucleic acid “colonies”are described, for example, in WO 98/44152, WO 98/44151, WO 00/18957 andWO 03/074734.

The invention may be understood with reference to the following exampleswhich are to be understood as illustrative, and not limiting, of thepresent invention.

Preparation 1: Synthesis of N-(5-bromoacetamidylpentyl) Acrylamide(BRAPA)

N-Boc-1,5-diaminopentane toluene sulfonic acid was obtained fromNovabiochem. The bromoacetyl chloride and acryloyl chloride wereobtained from Fluka. All other reagents were Aldrich products.

To a stirred suspension of N-Boc-1,5-diaminopentane toluene sulfonicacid (5.2 g, 13.88 mmol) and triethylamine (4.83 ml, 2.5 eq) in THF (120ml) at 0° C. was added acryloyl chloride (1.13 ml, 1 eq) through apressure equalized dropping funnel over a one hour period. The reactionmixture was then stirred at room temperature and the progress of thereaction checked by TLC (petroleum ether ethyl acetate 1:1). After twohours, the salts formed during the reaction were filtered off and thefiltrate evaporated to dryness. The residue was purified by flashchromatography (neat petroleum ether followed by a gradient of ethylacetate up to 60%) to yield 2.56 g (9.98 mmol, 71%) of product 2 as abeige solid. ¹H NMR (400 MHz, d₆-DMSO): 1.20 1.22 (m, 2H, CH₂),1.29-1.43 (m, 13H, tBu, 2×CH₂), 2.86 (q, 2H, J=6.8 Hz and 12.9 HZ, CH₂),3.07 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂), 5.53 (dd, 1H, J=2.3 Hz and 10.1Hz, CH), 6.05 (dd, 1H, J=2.3 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 HZ, CH), 6.77 (t, 1H, J=5.3 Hz, NH), 8.04 (bs, 1H, NH). Mass(electrospray+) calculated for C₁₃H₂₄N₂O₃ 256. found 279 (256+Na⁺).

Product 2 (2.56 g, 10 mmol) was dissolved in trifluoroaceticacid:dichloromethane (1:9, 100 ml) and stirred at room temperature. Theprogress of the reaction was monitored by TLC (dichloromethane:methanol9:1). On completion, the reaction mixture was evaporated to dryness, theresidue co-evaporated three times with toluene and then purified byflash chromatography (neat dichloromethane followed by a gradient ofmethanol up to 20%). Product 3 was obtained as a white powder (2.43 g, 9mmol, 90%). ¹H NMR (400 MHz, D20): 1.29-1.40 (m, 2H, CH₂), 1.52 (quint.,2H, J=7.1 Hz, CH₂), 1.61 (quint., 2H, J=7.7 Hz, CH₂), 2.92 (t, 2H, J=7.6Hz, CH₂), 3.21 (t, 2H, J=6.8 Hz, CH₂), 5.68 (dd, 1H, J=1.5 Hz and 10.1Hz, CH), 6.10 (dd, 1H, J=1.5 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH). Mass (electrospray+) calculated for C₈H₁₆N₂O 156.found 179 (156+Na⁺).

To a suspension of product 3 (6.12 g, 22.64 mmol) and triethylamine(6.94 ml, 2.2 eq) in THF (120 ml) was added bromoacetyl chloride (2.07ml, 1.1 eq), through a pressure equalized dropping funnel, over a onehour period and at −60° C. (cardice and isopropanol bath in a dewar).The reaction mixture was then stirred at room temperature overnight andthe completion of the reaction was checked by TLC(dichloromethane:methanol 9:1) the following day. The salts formedduring the reaction were filtered off and the reaction mixtureevaporated to dryness. The residue was purified by chromatography (neatdichloromethane followed by a gradient of methanol up to 5%). 3.2 g(11.55 mmol, 51%) of the product 1 (BRAPA) were obtained as a whitepowder. A further recrystallization performed in petroleum ether:ethylacetate gave 3 g of the product 1. ¹H NMR (400 MHz, d₆-DMSO): 1.21-1.30(m, 2H, CH₂), 1.34-1.48 (m, 4H, 2×CH₂), 3.02-3.12 (m, 4H, 2×CH2), 3.81(s, 2H, CH2), 5.56 (d, 1H, J=9.85 Hz, CH), 6.07 (d, 1H, J=16.9 Hz, CH),6.20 (dd, 1H, J=10.1 Hz and 16.9 Hz, CH), 8.07 (bs, 1H, NH), 8.27 (bs,1H, NH). Mass (electrospray+) calculated for C₁₀H₁₇BrN₂O₂276 or 278.found 279 (278+H⁺), 299 (276+Na⁺).

Preparation 2: Preparation of Glass Support

A: Cleaning of glass slides—The glass slides used for the preparation ofthe hydrogel surfaces were cleaned using the following in-houseprotocol: the slides were sequentially incubated in Decon TM, 1M aqueoussodium hydroxide and finally 0.1 M hydrochloric acid (aq). After eachstep, the slides were sonicated in MilliQ H₂O. The cleaned slides werestored in ethanol.

B: Binder silanization of the glass slides (optional)—The cleaned glassslides were baked at 120° C. for two hours prior to silanization. Aftercooling down in a dessicator filled with argon, the slides wereincubated at room temperature in a 2% v/v solution of either3-(trimethoxysilyl)propyl methacrylate or (3-acryloxypropyl)trimethoxysilane in toluene HPLC grade. The slides were then rinsed carefully withtoluene and cured for two hours at 120° C. The silanized slides werestored in a dessicator filled with argon.

Preparation 3: Preparation of the Polymerisation Mixture

Acrylamide (Purity 99+%, 0.4 g) was dissolved in MilliQ H₂O (10 mls)(Solution I). Potassium or ammonium persulfate (0.25 g) was dissolved inMilliQ H₂O (5 ml) (Solution II). N-(5-bromoacetamidylpentyl)acrylamide(BRAPA) (33 mg) was dissolved in DMF (330 pI) (Solution III).

Solution I was degassed 10 minutes with argon. Solution III was added tosolution I.

Example 1: Synthesis of Polyacrylamide-Based Surface Application of thePolyacrylamide Hydrogel to the Glass Slides

Method I—Two slides (optionally silanised) were assembled with a silicongasket in between to form a polymerisation cell. The slides and thegasket were held together with binder clips. Polymerisation mixture (800pI) was injected in each polymerisation cell. The polymerisationproceeded at room temperature' for 1.5 hr. The polymerisation cells werethen disassembled and the slides washed thoroughly under running MilliQH₂O. The slides were then dried and stored under argon.

Method II—Slides (optionally silanised) were put into a clean coplinjar. The polymerisation mixture was poured into the jar to cover theslides. The polymerisation proceeded at room temperature for 1.5 hr. Theslides were then removed from the coplin jar one by one and rinsed underrunning MilliQ H₂O. The slides were then introduced in clean plasticvials containing MilliQ H₂O and vortexed for 20 seconds. The slides wererinsed with running MilliQ H₂O, dried and stored under argon.

Example 2: Immobilisation of Polynucleotides on Polyacrylamide Surfaces

The following constitutes representative procedures for theimmobilisation of 5′-phosphorothioate-modified polynucleotides to thenew surface. Oligos with a 3′-fluorescent label are typically used forappraising fundamental surface characteristics.

A: Bulk Application (Suitable for Microarrays)

Polynucleotide (1 μM) in the printing buffer (potassium phosphate 100mM, pH 7) was applied to the surface as 1 μl drops. The slide was thenplaced in a humid chamber at room temperature for 1 hour. The printedslide was then rinsed with MilliQ H₂O and vortexed in hot washing buffer(Tris HCl 10 mM, EDTA 10 mM, pH 8 (at 80-90° C.)). The printed slide wasfinally rinsed with MilliQ H₂O, dried under a flow of argon and storedin the dark before imaging.

To immobilise polynucleotides over a large area, a gasket was placedonto the slide, the polynucleotide solution injected into the chamberformed and a cover slip placed over the gasket. The slide was thenincubated for 1 h at room temperature in a humid chamber and processedas described above.

B: Single Molecule Array Application

The coated slide was fitted into a custom-made flow cell. Polynucleotide(400 μl, 0.1 to 10 nM). in the printing buffer was injected into thecell. The concentration of polynucleotide is chosen to achieve a precisesingle molecule density of polynucleotides on the surface. The cell wasincubated in the dark at room temperature for 1 h. The printed surfacewas then washed by sequentially injecting printing buffer (20 ml), hotwashing buffer (20 ml, 80-90° C.) and finally MilliQ H₂O (20 ml).

Example 3: Thermal Stability of the Immobilised Polynucleotides

A. Bulk application (suitable for microarrays)—A printed slide wasimaged with a fluorescence scanner in the presence of a fluorescencereference control slide containing attached Cy3 dye. The printed slidewas then incubated in a jar containing printing buffer at a presettemperature in the dark. The slide was imaged at regular intervals andthe fluorescence intensity recorded. The fluorescence intensity of aspot is proportional to the amount of polynucleotide immobilised on thatarea. A plot of the variation of the fluorescence intensity with timegave a stability profile for attached polynucleotide on thepolyacrylamide surface.

B: Single molecule array application—A slide was printed in a flow cellas described above. Printing buffer is injected through the cell at arate of 1 ml/min, at a preset temperature, and the slide imaged atregular intervals using a custom-made total internal reflectionfluorimeter instrument. The stability profile of the single moleculearray is obtained by plotting the variation of the number of singlemolecules counted in a specific area with time.

Example 4: Evaluation of Surface Passivation Towards Nucleotide Stickingat SMA Level

A coated slide was fitted into a custom-made flow cell. The cell wasflushed with MilliQ H₂O (10 ml) then phosphate printing buffer (0.1 M,pH 7.0) and then incubated for 30 minutes at room temperature. Thisprocedure constituted a mock DNA couple to the surface. The cell wasflushed with MilliQ H₂O (10 ml), hot TE buffer (10 ml, 10 mM Tris.HCl,10 mM EDTA, pH 8.0) then MilliQ H₂O (10 ml). The slide was then imagedusing a custom-made total internal reflection fluorimeter instrument togive a fluorescent background reading. The cell was then flushed withenzymology buffer (10 ml, 50 mM Tris.HCl, 4 mM MgSO₄, 0.2 mM MnCl₂,0.05% Tween 20, pH 8.0). A solution of fluorescently-labelled nucleotide(400 μl, 0.2 to 2 μM in enzymology buffer) was then injected into thecell and the cell incubated at a preset temperature for 30 minutes. Thecell was flushed with wash buffer (10 ml, 50 mM Tris.HCl, 4 mM MgSO₄,0.05% Tween 20, pH 8.0), high salt buffer (10 ml, 50 mM Tris.HCl, 1MNaCl, 4 mM MgCl₂, 0.05% Tween 20, pH 8.0), TE buffer (10 ml, compositionas above) and MilliQ H₂O (10 ml). The slide was then imaged using acustom-made total internal reflection fluorimeter instrument todetermine the level of nucleotide sticking to the surface.

Example 5: Evaluation of Enzymology on Surface Immobilised Hairpin DNA

A: Single molecule array application—A coated slide was fitted into acustom-made flow cell. A solution of self-priming DNA hairpin (200 μl, 1nM, 0.1M KPi, pH 7.0) was then injected into the flow cell and the cellincubated for 1 hr at room temperature. The cell was then flushed withboiling TE buffer (20 ml, 10 mM Tris.HCl, 10 mM EDTA, pH 8.0) and MilliQH₂O (20 ml). The slide was then imaged using a custom-made totalinternal reflection fluorimeter instrument to give a fluorescentbackground reading. The flow cell was then flushed with wash buffer (20ml, 50 mM Tris.HCl, 4 mM MgSO₄, 0.05% Tween 20, pH 8.0) and thenenzymology mix (2×100 μl, 0.2 mM fluorescently-labelled nucleotide, 5μg/ml DNA polymerase, 50 mM Tris.HCl, 4 mM MgSO₄, 0.4 mM MnCl₂, 0.05%Tween 20, pH 8.0) was injected into the flow cell. The flow cell wasincubated at 45° C. for 30 minutes. The flow cell was then flushed at aflow rate of 0.5 ml/s with wash buffer (20 ml, 50 mM Tris.HCl, 4 mMMgSO₄, 0.05% Tween 20, pH 8.0), high salt wash buffer (20 ml, 50 mMTris.HCl, 1 M NaCl, 4 mM MgSO₄, 0.05% Tween 20, pH 8.0), TE buffer (20ml, 10 mM Tris.HCl, 10 mM EDTA, pH 8.0) and milliQ H₂O (20 ml). Theslide was then imaged using a custom-made total internal reflectionfluorimeter instrument to determine the level of enzyme incorporation ofthe fluorescently-labelled nucleotide into the hairpin.

Example 6: Preparation of a Polyelectrolyte-Treated FusedSilica-Supported Hydrogel and a Demonstration that it is More PassiveTowards Functionalised Labelled Nucleotides when Compared to aNon-Treated Control

A: A polyacrylamide-based glass-supported hydrogel is prepared asdescribed in Example 1.

B: Pretreatment with polyallylamine hydrochloride followed bypolyacrylic acid. Treatment of a polyacrylamide hydrogel comprising 1mol % BRAPA, as described in Part A, is effected by contacting thehydrogel with a solution of polyallylamine hydrochloride (2 mg/ml)MilliQ H₂O at pH 8. Contacting is effected for 30 min at roomtemperature after which the solution is treated with polyacrylic acid (2mg/ml MilliQ H₂O at pH 8.2). The solution is incubated for 30 min atroom temperature followed by treatment with MilliQ H₂O. The surfacetreated with the two layers of polyelectrolyte demonstrates a reductionin sticking of fluorescently functionalised nucleotides when compared toa control hydrogel surface not treated with the polyelectrolytes.

Example 7: Demonstration that of a Poly(Ethylene Glycol)-Treated FusedSilica-Supported Hydrogel is More Passive Towards FunctionalisedLabelled Nucleotides when Compared to a Non-Treated Control

Example 6 was repeated except that instead of treatment withpolyallylamine hydrochloride following by polyacrylic acid, the hydrogelprepared as in example 1 is treated with poly(ethylene glycol) 8000(Sigma). The surface treated with the poly(ethylene glycol) 8000demonstrates a reduction in sticking of fluorescently functionalisednucleotides when compared to a control hydrogel surface not treated withthe poly(ethylene glycol) 8000.

Example 8: Demonstration that Treatment of a Polyelectrolyte- orPoly(Ethylene Glycol)-Treated Fused Silica-Supported Hydrogel-BasedMolecular Array is More Passive Towards Functionalised LabelledNucleotides when Compared to a Non-Treated Control

Examples 6 and 7 are repeated except that instead of applying thepolyelectrolytes or poly(ethylene glycol) to a fused silica-supportedhydrogel as such, instead an array of polynucleotides is treated and thearrays so modified used in sequencing reactions with fluorescentlylabelled nucleotides. The surfaces treated with either the two layers ofpolyelectrolyte, or the poly(ethylene glycol) 8000, demonstrate areduction in sticking of fluorescently functionalised nucleotides whencompared to a control hydrogel surfaces not so treated.

Example 9: Preparation of Plastics-Supported Hydrogel

Plastic substrates were obtained from Amic and were cleaned with Decon90 overnight. These were poly(methyl methacrylate) (Amic PMMA), andcyclic olefin 1060 and 1420 plastics (Amic (COP) 1060 and Amic (COP)1420). The next day they were rinsed extensively with MilliQ water anddried. A 2% w/v acrylamide solution was made up by dissolving 1.3 gacrylamide in 65 ml of MilliQ water. This solution was then purged withargon for 15 minutes to remove oxygen, which may inhibit thepolymerisation reaction. BRAPA (107 mg) was then dissolved with 1.07 mlof dimethylformamide (DMF) and added to the degassed acrylamidesolution. After mixing, 75 μl of TEMED catalyst was added to theacrylamide/BRAPA solution. Then polymerisation was started by adding0.65 ml of a 0.05 g/ml solution of potassium persulfate initiator to theacrylamide/BRAPA/TEMED solution. The acrylamide/BRAPA/TEMED/persulfatesolution was quickly mixed and added to a coplin jar containing theclean dry plastic (and glass) substrates. After 90 minutes the slideswere removed from the polymerising mixture and rinsed copiously withMilliQ under flow. They were then vortexed 20 seconds in MilliQ andrinsed a second time under flow before being dried with Argon. Slidestreated in this way are hereinafter referred to as being slides treatedwith silane-free acrylamide (SFA) or “support with SFA”.

Examples 10: Functionalisation of Plastics-Supported Hydrogel

Following slide preparation as described in Example 9, CultureWellcoverglass gaskets from GraceBio labs were attached to the slides for 1hour. 1 μM phosphorothioate-Cy3-DNA (positive) and 1 μM hydroxyl-Cy3-DNA(negative, control DNA) in 10 μM phosphate buffer pH 7 were then spottedin wells on the plastic (and glass) slides and coupling carried out for1 hour in a humidity chamber at room temperature (20° C.). Followingcoupling each slide was rinsed copiously with higher ionic strength 0.10M phosphate buffer. The gasket was then carefully removed and the slidethen vortexed 20 seconds in 10 mM Tris/10 mM EDTA pH 8 buffer. Finallythe slide was rinsed with MilliQ water under flow, then dried withargon.

Example 11: Detection of Oligonucleotide Functionalisation ofPlastics-Supported Hydrogel

Fluorescence scanning was performed on a Typhoon 8600 imager at 550V,100 μm resolution in the Cy3 channel (532 laser excitation).

The images shown in FIG. 1 show the attempted coupling of positive (PS,phosphorothioate) and negative (OH, hydroxyl control) DNA to Amic PMMAand 1060 and 1420 plastics with and without SFA. As may be seen bycomparison of the 1st and 2nd columns of images, a fluorescent signal isonly obtained when the plastic has been first coated with SFA. Somenon-specific binding of 1 μM hydroxyl DNA is observed but less than inthe case of the glass substrate. This suggests that substrate effectsare still present despite the presence of SFA coating.

The relative levels of positive signal due to phosphorothioate bindingare shown in FIG. 2 along with the measured signal-to-noise values(phosphorothioate DNA: hydroxyl DNA). The graph shown in FIG. 3 showsthat the apparent stability of the specifically adsorbedphosphorothioate DNA in 50 mM phosphate buffer pH 7 at 65° C. isessentially the same as that for glass. Approximately 40% of thestarting signal is left after 7 days incubation in 50 mM phosphate pH 7at 65° C. This value is slightly lower than expected for both glass andplastic substrates and is attributed to the fact that the samples werealways scanned dry after briefly rinsing with MilliQ water. When dry,the fluorescent dye is greatly affected by environmental conditionsparticularly ozone levels.

Example 12: Further Preparation of Plastics-Supported Hydrogels

Other plastics (e.g. polystyrene) and plastics of the same type but fromdifferent suppliers were also tested and hybridisation studies attemptedas follows:

Polystyrene from Corning, Zeonex E48R (a cyclic olefin polymer) fromZeon Chemicals Ltd. and poly(methyl methacrylate) from the TechnicalUniversity of Denmark, as well as Spectrosil glass substrates, werecleaned as described in Example 9. Some clean samples were kept asidewhile others were coated with SFA as described in Example 9.

1 μM phosphorothioate-Cy3-DNA (positive), 1 μM hydroxyl-Cy3-DNA(negative, control DNA), 1 μM unlabeled P5 primer with a 10T spacer and1 μM unlabeled P7 primer with a 10T spacer were coupled to substrates asprepared in Example 12, and cleaned but not treated with SFA. Scanningwas then performed as described in Example 12. Following scanninghybridisation was carried out using a Texas Red labelled complementarytarget to the P5 primer (P5′) by attaching a square silicone gasket tothe substrate then placing another clean (uncoated glass) slide on topto form a cell. An injection of 0.5 μM complementary target in 5× sodiumcitrate (SSC), 0.1% Tween 20 buffer pH 7 was then made into the spacecreated by the gasket and the cell heated in an oven to 95° C. for 30minutes. The oven temperature was then switched to 50° C. and the cellallowed to cool for 2 hours. Afterwards the cell was removed and allowedto cool at room temperature for 10 minutes in the dark. Thecomplementary target solution was then removed with a syringe and fresh5×SSC, 0.1% Tween 20 buffer at room temperature injected, then removed.This washing procedure was repeated 5 further times. The cell was thendismantled and the substrate agitated in a beaker of fresh 5×SSC, 0.1%Tween 20 for 2 minutes. The substrate was then transferred to a beakercontaining higher stringency (0.1×SSC, 0.1% Tween 20) and agitated for 2minutes. This was repeated once more then the substrate dried withargon. The samples were then scanned again this time at 700 V in the Roxchannel using 633 nm excitation (100 μm resolution).

The images shown in FIG. 4 shows below the coupling of the Cy3-labelledphosphorothioate (GW2) and hydroxyl DNA (GW4) to glass and plasticsubstrates with and without SFA coatings. Again, the same results wereobtained (i.e. coupling of phosphorothioate DNA only when SFA ispresent) although the signal intensities and signal-to-noise ratios varyslightly from before (see graph).

Example 13: Formation of Clustered Arrays by Template Hybridisation andPCR Overview:

The inventors' approach involves an initial step of preparing thesurface of the solid support, followed by covalent attachment (grafting)of primers to generate a surface ready for use in PCR. The PCR templatemay then be hybridised to attached primers immediately prior to the PCRreaction. The PCR reaction thus begins with an initial primer extensionstep rather than template denaturation. This approach is illustrated inFIG. 6.

Experimental:

The solid supports used in this experiment were 8-channel glass chipssuch as those provided by Micronit (Twente, Nederland) or IMT(Neuchatel, Switzerland). However, the experimental conditions andprocedures are readily applicable to other solid supports.

Chips were washed as follows: neat Decon for 30 min, milliQ H₂O for 30min, NaOH 1N for 15 min, milliQ H₂O for 30 min, HCl 0.1N for 15 min,milliQ H₂O for 30 min.

Chips were then coated with polyacrylamide hydrogel as described inExample 1.

5′-phosphorothioate oligonucleotides were grafted onto the surface ofthe hydrogel in 10 mM phosphate buffer pH7 for 1h at RT. The followingexemplary primers were used:

P7 primer with polyT spacer:

(SEQ ID NO: 1) 5′phosphorothioate-TTTTTTTTTTCAAGCAGAAGACGGCATACG A-3′

P5 primer with polyT spacer:

(SEQ ID NO: 2) 5′phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCGA- 3′

Primers were used at a concentration of 0.5˜M in the grafting solution.

The template used was a fragment of pBluescript T246 containingsequences complementary to the P7/P5 primers shown above. Thehybridization procedure began with a heating step in a stringent buffer(95° C. for 5 minutes in TE) to ensure complete denaturation prior tohybridisation of the template. Hybridization was then carried out in5×SSC, using template diluted to a final concentration of 5 nM. Afterthe hybridization, the chip was washed for 5 minutes with milliQ waterto remove salts.

Surface amplification was carried out by thermocycled PCR in an MJResearch thermocycler.

A typical PCR program is as follows:

1—97.5° C. for 0:45

2—X° C. for 1:30

3—73° C. for 1:30

4—Goto 1 [40] times

5—73° C. for 5:00

6—20° C. for 3:00

7—End

Since the first step in the amplification reaction is extension of theprimers bound to template in the initial hybridisation step the firstdenaturation and annealing steps of this program were omitted (i.e. thechip was placed on the heating block only when the PCR mix was pumpedthrough the channels and the temperature was 73° C.).

The annealing temperature (X° C., step 2) depends on the primer pairthat is used. Experiments have determined an optimal annealingtemperature of 57° C. for P5/P7 primers.

For other primer-pairs the optimum annealing temperature can bedetermined by experiment. The number of PCR cycles may be varied ifrequired.

PCR was carried out in a reaction solution comprising 1×PCR reactionbuffer (supplied with the enzyme) 1M betaine, 1.3% DMSO, 200 μM dNTPsand 0.025 U/μL Taq polymerase.

In order to visualise colonies the glass chips were stained with SYBRGreen-I in TE buffer (1/10 000) and then viewed using an epifluorescencemicroscope. It was observed that amplified colonies had formed inchannels treated as described above, but not in control channels inwhich the template was added at the primer grafting stage (data notshown).

Example 14: Effect of Spacer Length on Polynucleotide HybridisationBackground

DNA microarrays may be used to carry out thousands of heterogeneous(solid-liquid interface) hybridisations simultaneously to determine geneexpression patterns or to identify genotype. Hybridisation on thesearrays depends on a number of factors including probe density (Peterson,A. W. et al. (2001) Nucl. Acids Res. 29, 5163-5168) while kinetic ratesand equilibrium binding constants may differ markedly from the solutionphase.

Due to steric hindrance and a reduced degree of freedom the proximity tothe support surface is a key criterion affecting hybridisation yield(Weiler, J et al. (1997) Nucl. Acids Res. 25, No. 14, 2792-2799).Shchepinov et al., determined that 60 atoms was the optimal spacerlength for a hydrophilic PEG-phosphoroamidite synthon spacer(Shchepinov, M. S. et al. (1997) Nucl. Acids Res. 25, 1155-1161). Thisresulted in a 50-fold increase in hybridisation yield. Above 60 atoms(approx. 10 glycol units) hybridisation yield decreased until at 30units the yield of hybridisation was the same as that with no spacer atall. Moreover, the yield of hybridisation was affected by the chargedensity along the spacer while being independent of charge type.

The hybridisation studies outlined below show that it was possible toimprove hybridisation yield by incorporating a polyT spacer into theprimer. Similar oligoT spacers have been shown previously to produce a20-fold enhancement of hybridization (Guo, Z. et al. (1994) Nucl. AcidsRes. 22, 5456-5465).

Experimental:

(1) Hybridization on a Microplate-Effect of Spacer

Modification of Glass with Silane-Free Acrylamide (SFA):

A 96 well, glass bottom, polystyrene microplate was placed into Decon 90overnight. The next day the microplate was rinsed extensively withMilliQ water, then dried with Argon. The glass surface of each well ofthe microplate was then coated with silane-free acrylamide (SFA).Briefly, acrylamide was dissolved in water to give a 2% w/v solution andargon then bubbled through this solution for 15 minutes.

82.5 mg of BRAPA (the active monomer) was dissolved in 0.825 mldimethylformamide (DMF). This solution was then added to 50 ml ofacrylamide solution to give a 2 mol % BRAPA solution with respect toacrylamide. After mixing 57.5 μl TEMED was added to the acrylamide/BRAPAsolution. A potassium persulfate solution was then prepared bydissolving 0.1 g in 2 ml MilliQ water. 0.5 ml of the initiator solutionwas then added to the degassed acrylamide/BRAPA/TEMED solution and,after mixing, 0.4 ml of the polymerisation mixture was pipetted intoeach well of the microplate. Polymerisation was allowed to proceed for1.5 hours, then the microplate was washed on an automated microplatewasher with program AHPEM160. Following washing the plate was driedunder argon and stored overnight under vacuum.

Coupling of Oligo Primers to SFA Surface:

The following 4 primers were coupled to the surface at 2.0 μM, 1.0 μM,0.5 μM and 0.1 μM concentrations from 10 μM phosphate buffer pH 7:

1) P7 primer without polyT spacer:

(SEQ ID NO: 3) 5′phosphorothioate-CAAGCAGAAGACGGCATACGA-3′

2) P7 primer with polyT spacer:

(SEQ ID NO: 1) 5′phosphorothioate-TTTTTTTTTTCAAGCAGAAGACGGCATACG A-3′

3) P5 primer without polyT spacer:

(SEQ ID NO: 4) 5′phosphorothioate-AATGATACGGCGACCACCGA-3′

4) P5 primer with polyT spacer:

(SEQ ID NO: 2) 5′phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCGA- 3′

Coupling was carried out using 0.1 ml oligonucleotide solution for 1hour in a humid environment at room temperature. Afterwards themicroplate was washed with 0.10 M phosphate buffer pH 7 on an automatedmicroplate washer.

Hybridisation:

Hybridisation to the primers (FIG. 7) was carried out using thefollowing complementary Texas Red labelled targets:

P5′ complementary target to P5 sequence:

(SEQ ID NO: 5) 5′Texas Red-TCGGTGGTCGCCGTATCATT-3′

P7′ complementary target to P7 sequence:

(SEQ ID NO: 11) 5′Texas Red-TCGTATGCCGTCTTCTGCTTG-3′

The target volume was 0.1 ml and the concentration was 0.5 μM. Thetarget was made up in two different hybridisation buffers and tested.The composition of the TAQ PCR buffer was 1M betaine, 1.3% DMSO, 10 mMTris, 1.5 mM MgCl₂ and 50 mM KCl (pH 9). The other buffer contained5×SSC (diluted from a 20× stock) and 0.1% (v/v) Tween 20 (pH 7). Aspecial PCR film was used to seal the wells of the microplate andprevent evaporation on heating. The plate was then placed on a PCR blockwith lid and submitted to the following conditions:

1) 0.5° C. to 97.5° C.

2) 97.5° C. for 2 mins 30 secs

3) 97.5° C. for 2 sees −0.1° C. per cycle

4) Goto 3, 574 times

5) 40.0° C. for 15 mins

6) End

After heating and cooling the plate was washed 15 times using themicroplate washer (modified program ‘AHPEM170’) with 5×SSC, 0.1% v/vTween 20 pH 7, then 6 times using the same program but with 0.1×SSC,0.1% v/v Tween 20 pH 7. Following washing the plate was scanned wetunder on a Typhoon 9600 imager at 700V with the ROX filter, 633 nmexcitation, 200 mM pixel size.

Results:

The results presented in FIG. 8 show a clear improvement inhybridisation signal for both the P5 and P7 primer upon adding a spacerwith 10 T bases. Not only is the signal much higher but thesignal-to-noise (specific to non-specific hybridisation) also improvesimmensely.

(2) Hybridization on Typhoon Slides —Effect of Spacer Coupling of OligoPrimers to SFA Surface:

The following 4 primers were coupled to the surface at 1.0 μMconcentrations from 10 mM phosphate buffer pH 7:

1) P7 primer without polyT spacer:

(SEQ ID NO: 3) 5′phosphorothioate-CAAGCAGAAGACGGCATACGA-3′2) P7 primer with polyT spacer:

(SEQ ID NO: 1) 5′phosphorothioate-TTTTTTTTTTCAAGCAGAAGACGGCATACG A-3′3) P5 primer without polyT spacer:

(SEQ ID NO: 4) 5′phosphorothioate-AATGATACGGCGACCACCGA-3′4) P5 primer with polyT spacer:

(SEQ ID NO: 2) 5′phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCGA- 3′

Coupling was carried out for 1 hour by spotting 7 μl of each oligo intoone or more wells created by sticking a Grace Biolab CultureWellcoverglass gasket (gasket 1, FIG. 9) onto a Typhoon slide previouslymodified with 2% acrylamide, 2 mol % BRAPA silane-free acrylamide.During coupling the slides were kept in the dark in a humidity chamber.After coupling the slides were rinsed with 250 ml of 0.1 M phosphatebuffer pH 7 from a wash bottle. Then each slide was vortexed 20 secondsin 10 mM/10 mM Tris/EDTA pH 8 buffer, then rinsed with MilliQ water anddried.

Hybridisation:

Hybridisation to the primers (FIG. 7) was carried out using thefollowing complementary Texas Red labelled targets:

P5′ complementary target to P5 sequence:

(SEQ ID NO: 5) 5′Texas Red-TCGGTGGTCGCCGTATCATT-3′

P7′ complementary target to P7 sequence:

(SEQ ID NO: 11) 5′Texas Red-TCGTATGCCGTCTTCTGCTTG-3′

A silicone gasket (gasket 2, FIG. 9) was attached to the primer modifiedslide and a clean glass slide placed on top to create a sealed chamber.Clips were used to ensure sealing. The space created by the gasket wasthen filled with one of the complementary targets (P5′ or P7′) then theprimer-modified side was placed in contact with an aluminium heatingblock. A box was placed on top to prevent access of light. Thetemperature of the aluminium block was increased from room temperatureto 95° C. (required 15 minutes). The temperature of the heater was thenturned down to 40° C. and the slides allowed to cool to 50° C. (required2 hours). Once at 50° C. the slide was allowed to cool to roomtemperature during 10 minutes. The complementary target solution wasthen removed with a syringe and the inside washed by injection 6× with5×SSC, 0.1% Tween 20. The gasket, clips and slides were then dismantledand the primer-modified slide rinsed with agitation in a tube containing5×SSC, 0.1% Tween 20 for 2 minutes. This was repeated then theprimer-modified slide was rinsed with 0.1×SSC, 0.1% Tween 20, twice for2 minutes with agitation. The slide was then dried with Argon andscanned dry at 700V as above.

Results: The results presented in FIGS. 10 and 11 show a clearimprovement in hybridisation signal for both the P5 and P7 primer uponadding a spacer with 10 T bases. Not only is the signal much higher butthe signal-to-noise (specific to nonspecific hybridisation) alsoimproves immensely.

(3) Hybridization on Typhoon Slides —Effect of Spacer Length Coupling ofOligo Primers to SFA Surface:

The following 8 primers were coupled to the surface at 1.0 μMconcentrations from 10 mM phosphate buffer pH 7:

1) P7 primer without polyT spacer:

(SEQ ID NO: 3) 5′phosphorothioate-CAAGCAGAAGACGGCATACGA-3′2) P7 primer with polyT spacer:

(SEQ ID NO: 1) 5′phosphorothioate-TTTTTTTTTTCAAGCAGAAGACGGCATACG A-3′3) P5 primer without polyT spacer:

(SEQ ID NO: 4) 5′phosphorothioate-AATGATACGGCGACCACCGA-3′4) P5 primer with polyT spacer:

(SEQ ID NO: 2) 5′phosphorothioate-TTTTTTTTTTAATGATACGGCGACCACCG A-3′5) P5 primer with polyT spacer:

(SEQ ID NO: 7) 5′phosphorothioate-TTAATGATACGGCGACCACCGA-3′6) P5 primer with polyT spacer:

(SEQ ID NO: 8) 5′phosphorothioate-TTTTTAATGATACGGCGACCACCGA-3′7) P5 primer with polyT spacer:

(SEQ ID NO: 9) 5′phosphorothioate-TTTTTTTTTTTTTTTTTTTTAATGATACGGCGACCACCGA-3′8) P5 primer with C18 spacer:

(SEQ ID NO: 10) 5′phosphorothioate-C18-AATGATACGGCGACCACCGA-3′

Coupling was carried out for 1 hour by spotting 7˜l of eacholigonucleotide into one or more wells created by sticking a GraceBiolab CultureWell coverglass gasket (gasket 1, scheme 2) onto a Typhoonslide previously modified with 2% acrylamide, 2 mol % BRAPA silane-freeacrylamide. During coupling the slides were kept in the dark in ahumidity chamber. After coupling the slides were rinsed with 250 ml of0.1 M phosphate buffer pH 7 from a wash bottle. Then each slide wasvortexed 20 seconds in 10 mM/10 mM Tris/EDTA pH 8 buffer, then rinsedwith MilliQ water and dried.

Hybridisation:

Hybridisation to the primers (FIG. 7) was carried out using thefollowing complementary Texas Red labelled targets:

P5′ complementary target to P5 sequence:

(SEQ ID NO: 5) 5′Texas Red-TCGGTGGTCGCCGTATCATT-3′

P7′ complementary target to P7 sequence:

(SEQ ID NO: 11) 5′Texas Red-TCGTATGCCGTCTTCTGCTTG-3′

Hybridisation was carried out as above. A silicone gasket (gasket 2,FIG. 9) was attached to the primer modified slide and a clean glassslide placed on top to create a sealed chamber. Clips were used toensure sealing. The space created by the gasket was then filled with oneof the complementary targets (P5′ or P7′) then the primer-modified sidewas placed in contact with an aluminium heating block. A box was placedon top to prevent access of light. The temperature of the aluminiumblock was increased from room temperature to 95° C. (required 15minutes). The temperature of the heater was then turned down to 40° C.and the slides allowed to cool to 50° C. (required 2 hours). Once at 50°C. the slide was allowed to cool to room temperature during 10 minutes.The complementary target solution was then removed with a syringe andthe inside washed by injection 6× with 5×SSC, 0.1% Tween 20. The gasket,clips and slides were then dismantled and the primer-modified sliderinsed with agitation in a tube containing 5×SSC, 0.1% Tween 20 for 2minutes. This was repeated then the primer-modified slide was rinsedwith 0.1×SSC, 0.1% Tween 20, twice for 2 minutes with agitation. Theslide was then dried with Argon and scanned dry at 700V as above.

Results:

The results presented in FIG. 12 show that hybridisation yield increasessharply with polyT spacer length up to 10 Ts but between 10T and 20Tbases it begins to decrease. A reduced hybridisation yield above anoptimum spacer length has also been reported elsewhere (Shchepinov, M.S. et al. (1997) Nucl. Acids Res. 25, 1155-1161; Guo, Z. et al (1994)Nucl. Acids Res. 22, 5456-5465). One possible explanation could be thatlarger primers have lower primer density on the surface and thereforeresult in a smaller hybridisation signal. A C18 spacer results in only asmall improvement in hybridisation yield suggesting that spacerhydrophilicity is also an important factor.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments, it will be understood by those skilled in the artthat various changes in form and details may be made without departingfrom the scope of the invention encompassed by the claims.

1. (canceled)
 2. A method of preparing an array of nucleic acids,comprising: immobilizing a population of polynucleotides on a surface ofa solid support, wherein the population of polynucleotides comprises afirst plurality of polynucleotides, wherein each polynucleotide of thefirst plurality of polynucleotides comprises the nucleotide sequence setforth in SEQ ID NO:03 or
 04. 3. The method of claim 2, wherein the firstplurality of polynucleotides comprises the nucleotide sequence set forthin SEQ ID NO:03, and the population of polynucleotides further comprisesa second plurality of polynucleotides, wherein each polynucleotide ofthe second plurality of polynucleotides comprises the nucleotidesequence set forth in SEQ ID NO:04.
 4. The method of claim 2, whereineach polynucleotide of the population of polynucleotides comprises apolyT spacer comprising from 2 to 10 thymine nucleotides.
 5. The methodof claim 2, wherein the population of polynucleotides are attacheddirectly to the surface of the solid support.
 6. The method of claim 2,wherein immobilizing the population of polynucleotides comprisesimmobilizing the population of polynucleotides to a hydrogel immobilizedon the surface of the solid support.
 7. The method of claim 6, whereinthe hydrogel has a thickness less than 100 nm.
 8. The method of claim 6,further comprising preparing the hydrogel by polymerizing a firstcomonomer with a second comonomer, wherein the first comonomer isselected from the group consisting of acrylamide, methacrylamide,hydroxyethyl methacrylate and N-vinyl pyrrolidinone, and wherein thesecond comonomer is selected from the group consisting of afunctionalized acrylamide, acrylate, methacrylate and methacrylamide. 9.The method of claim 6, wherein preparing the hydrogel comprisespolymerizing the acrylamide with N-(5-bromoacetamidylpentyl)acrylamide(BRAPA).
 10. The method of claim 2, wherein the solid support comprisesa material selected from the group consisting of a silica-basedcompound, plastic, gold, titanium dioxide, and silicon.
 11. The methodof claim 2, further comprising hybridizing a population of templatenucleic acids to the population of polynucleotides.
 12. The method ofclaim 11, wherein each template nucleic acid comprises a first endcapable of hybridizing to SEQ ID NO:03, and a second end, wherein thetemplate nucleic acids are different from each other.
 13. The method ofclaim 11, wherein each template nucleic acid comprises genomic DNA. 14.The method of claim 11, further comprising extending the population ofpolynucleotides hybridized to the population of template nucleic acids.15. The method of claim 14, further comprising removing the populationof template nucleic acids from the extended population ofpolynucleotides.
 16. The method of claim 14, further comprisingsequencing the extended population of polynucleotides.
 17. A method ofsequencing a population of template nucleic acids, comprising: (a)hybridizing the population of template nucleic acids to a population ofpolynucleotides immobilized on a surface of a solid support, wherein thepopulation of polynucleotides comprises a first plurality ofpolynucleotides, wherein each polynucleotide of the first plurality ofpolynucleotides comprises the nucleotide sequence set forth in SEQ IDNO:03 or 04; (b) extending the population of polynucleotides hybridizedto the population of template nucleic acids; and (c) sequencing theextended population of polynucleotides.
 18. The method of claim 17,wherein the first plurality of polynucleotides comprises the nucleotidesequence set forth in SEQ ID NO:03, and the population ofpolynucleotides further comprises a second plurality of polynucleotides,wherein each polynucleotide of the second plurality of polynucleotidescomprises the nucleotide sequence set forth in SEQ ID NO:04.
 19. Themethod of claim 17, wherein each template nucleic acid comprises a firstend capable of hybridizing to SEQ ID NO:03, and a second end, whereinthe template nucleic acids are different from each other.
 20. The methodof claim 17, wherein each template nucleic acid has a length greaterthan 300 nucleotides.
 21. The method of claim 17, wherein each templatenucleic acid comprises genomic DNA.